A novel method for L-methionine production catalyzed by the

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A novel method for L-methionine production catalyzed by the aminotransferase ARO8 from Saccharomyces cerevisiae Yiping Wu, Musu ZHA, Sheng Yin, Huaqing Yang, Julien Boutet, Robert Huet, Chengtao Wang, and Baoguo Sun J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01451 • Publication Date (Web): 28 May 2018 Downloaded from http://pubs.acs.org on May 28, 2018

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Journal of Agricultural and Food Chemistry

A novel method for L-methionine production catalyzed by the aminotransferase ARO8 from Saccharomyces cerevisiae

Yiping Wu†§⊥, Musu Zha†§⊥, Sheng Yin†§*, Huaqing Yang†§, Julien Boutet‡, Robert Huet‡, Chengtao Wang†§*, Baoguo Sun†§

†

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing

Technology & Business University, Beijing 100048, China. §

Beijing Engineering and Technology Research Center of Food Additives, Beijing

Technology & Business University, Beijing 100048, China. ‡

Adisseo France SAS, Antony Parc 2, 10 Place du Général de Gaulle, F-92160

Antony, France; Bluestar Adisseo Nanjing Co., LTD, 389 Changfenghe Road, Nanjing Chemical Industry Park, Jiangsu Province, Nanjing 210047, China.

*Corresponding Author Phone: 86-10-68985252. Fax: 86-10-68985252. E-mail: [email protected] (Sheng Yin); [email protected] (Chengtao Wang)

1

ACS Paragon Plus Environment


1

ABSTRACT

2

The aminotransferase ARO8 was proved to play an efficient role in conversion of

3

L-methionine into methionol via the Ehrlich pathway in Saccharomyces cerevisiae in

4

our previous work. In this work, the reversible transamination activity of ARO8 for

5

conversion of α-keto-γ-(methylthio) butyric acid (KMBA) into methionine was

6

confirmed in vitro. ARO8 was cloned from S. cerevisiae S288c and over-expressed

7

in Escherichia coli BL21. A 2-fold higher aminotransferase activity was detected in

8

the recombinant strain ARO8-BL21 and ARO8 was detected in the supernatant of

9

ARO8-BL21 lysate with IPTG induction by SDS-PAGE analysis. The recombinant

10

ARO8 was then purified and used for transforming KMBA into methionine. An

11

approximately 100% conversion rate of KMBA into methionine was achieved by

12

optimized enzymatic reaction catalyzed by ARO8. This work fulfilled L-methionine

13

biosynthesis catalyzed by the aminotransferase ARO8 using glutamate and KMBA,

14

which provided a novel method for L-methionine production by enzymatic catalysis

15

with the potential application prospect in industry.

16 17

KEYWORDS: Methionine, Aminotransferase, ARO8, Saccharomyces cerevisiae

18 19 20 21 22 2


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INTRODUCTION

24

Methionine is an important amino acid widely used in animal nutrition, food and

25

medicine industry

26

2016 and is expected to grow by 6% each year in the future. Currently, methionine is

27

mainly produced by chemical synthesis 5, which normally generates the racemic

28

mixture of L-methionine and undesirable D-methionine 6. In contrast, enzymatic

29

catalysis can produce optical pure L-methionine. A well-known industrial-operated

30

process for methionine production is the enzymatic conversion of N-acetyl

31

DL-methionine to the pure L-form 4, and L-methionine could also be produced by

32

microbial fermentation. But the main drawback is the very complex biosynthesis

33

pathway of methionine with manifold feedback inhibitions 4. Hence, more efforts

34

have been taken in developing new simple and efficient approaches for L-methionine

35

production using cheap raw materials.

1-4

. The market demand of methionine exceed 1 million tons in

36

In yeast, methionine can be transformed into the fusel alcohol methionol via the

37

Ehrlich pathway 7. The main steps involves transamination of methionine into

38

4-methylthio-2-oxobutyric acid (KMBA), decarboxylation of KMBA into methional,

39

and dehydrogenation of methional into methionol (Fig. 1) 7. If the aforesaid

40

transamination and decarboxylation reactions were reversible, the industrial bulk

41

chemical methional would be an alternative source for high-value L-methionine

42

biosynthesis via enzymatic catalysis.

43

Though dozens of transaminases and decarboxylases were reported to get

44

involved in the Ehrlich pathway 8, in our previous work, over-expression of the 3



45

transaminase ARO8 and decarboxylase ARO10 were proved to significantly enhance

46

conversion of methionine into methional and methionol in S. cerevisiae S288c 9.

47

Moreover, it’s noteworthy that the transaminase reaction catalyzed by ARO8 seems

48

to be bidirectional as the aminotransferase could act on not only the aromatic amino

49

acids but also their oxo-acid analogues when the eligible amino donor is present 10.

50

Therefore, in this work, the aminotransferase ARO8 was cloned from S. cerevisiae

51

and over-expressed in E. coli. The recombinant ARO8 protein was purified for the

52

reversible transamination activity assay, by which KMBA was transformed into

53

L-methionine. An approximately 100% conversion rate of KMBA into L-methionine

54

was achieved by optimizing the enzymatic reaction condition. The work provided a

55

novel L-methionine production method by enzymatic catalysis with the potential

56

application prospect in industry.

57 58

MATERIALS AND METHODS

59

Strains, plasmids and culture conditions

60

Strains and plasmids used in this work are listed in Table 1. E. coli strains were

61

cultured in Luria-Bertain (LB) medium at 37℃ with vigorous shaking. S. cerevisiae

62

strains were grown in yeast extract peptone dextrose (YPD) medium at 30℃ with

63

vigorous shaking. When needed, antibiotics were added at the following

64

concentration: 200 µg/mL ampicillin for E. coli, 200 µg/mL G418 for S. cerevisiae.

65 66

Aminotransferase activity assay for methionine biosynthesis 4


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For aminotransferase activity assay, the fresh cultures of S. cerevisiae and E. coli

68

were grown in YPD and LB media with vigorous shaking at 200 rpm for 24 h,

69

respectively. Cells were harvested by centrifugation at 4℃ for 5 min at 1000 g. The

70

cell pellet was resuspended in 2 mL of cold lysis buffer (50 mM potassium

71

phosphate, 2 mM EDTA, 2 mM DTT, and 0.1 mM PLP, pH 7.5)

72

were prepared by sonication at 0℃ for 10 min at 2 s intervals in a horn-type

73

sonicator (225 W output, SL-650D, Nanjing, China) 12. Cell debris were removed by

74

centrifugation at 4℃ for 5 min at 1000 g. The clear supernatant was used for

75

enzymatic activity assay immediately.

11

. Cell extracts

76

The reaction system containing 100 µL of the clear supernatant, 700 µL of lysis

77

buffer, 100 µL of KMBA (10 mM, CAS 51828-97-8, SIGMA, Saint Louis, USA)

78

and 100 µL of glutamate (10 mM ) was incubated at 30℃ for 20 min. The protein

79

concentration of each clear supernatant sample was determined using the BCA

80

Protein Assay Kit (TIANGEN, Beijing, China). The amount of methionine was

81

determined

82

derivatization with 2, 4-dinitrofluorobenzene (DNFB) as described by Li et al. 13 and

83

one unit of aminotransferase activity was defined as formation of 1 µM methionine

84

in 20 min. HPLC analysis conditions were as follows: Inertsil ODS-3 column

85

(4.6×250 mm, 5 µm, SHIMADZU, Shanghai, China); injection volume 20 µL; oven

86

temperature 23℃; flow rate 1 mL/min; the eluate was monitored at 360 nm. Mobile

87

phase A (50 mM sodium acetate buffer containing 0.1% triethylamine, pH 6.4) and

88

phase B (50% acetonitrile / 50% water solution) ran on an gradient elution program.

by

HPLC

(SHIMADZU,

Shanghai,

China)

5


with

pre-column


89 90

DNA manipulation techniques

91

Standard DNA manipulation techniques were performed as described by Sambrook

92

& Russell (2001)

93

genomic DNA Kit following the manufacturer's instructions (TIANGEN, Beijing,

94

China). Plasmid DNA from E. coli was isolated using the High-purity Plasmid

95

Miniprep Kit according to the manufacturer's instructions (TIANGEN, Beijing,

96

China). Construction of recombinant DNA using restriction enzymes and ligase was

97

conducted according to the supplier's instructions (TRANSGEN, Beijing, China).

98

Standard heat shock transformation method was used to introduce plasmids DNA to

99

E. coli BL21 (DE3) 14.

14

. Genomic DNA from yeast was extracted using the Yeast

100 101

Construction of the ARO8 expression vector

102

Based on the ARO8 gene (GenBank accession no. NM_001181067.1), specific

103

primers (F-ARO8: 5’-ATGACTTTACCTGAATCAAAAGAC-3’; R-ARO8: 5’-

104

CTATTTGGAAATACCAAATTCTTC-3’) were designed for PCR. The ARO8 gene

105

was amplified by PCR from the genomic DNA of S. cerevisiae S288c. Amplification

106

reaction was performed using Takara PrimerSRTAR® MAX DNA Polymerase

107

following the manufacture's recommendations (TAKARA, China). The amplicon

108

was purified and recovered using the TaKaRa MiniBEST DNA Fragment

109

Purification Kit following the manufacture's recommendations (TAKARA, China).

110

The purified amplicon was linked to the expression vector pEASY-Blunt E1 using 6


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pEASY-Blunt E1 Expression Kit following the manufacture's recommendations

112

(TRANSGEN, China), generating the recombinant vector pEASY-ARO8. The DNA

113

ligation mixture was transformed into E. coli Trans-T1 and transformants were

114

screened on LB agar plates containing 200 µg/mL ampicillin. The positive

115

recombinant plasmid was then sequenced and further analyzed with DNAMAN

116

software package and BLAST Program at NCBI against the GenBank database.

117 118

Induced expression of the recombinant ARO8 in E. coli

119

The recombinant plasmid pEASY-ARO8 was transformed into E. coli BL21 (DE3)

120

(TIANGEN, Beijing, China) and transformants were screened on LB agar plates

121

containing 200 µg/mL ampicillin. The recombinant strain BL21-ARO8 was

122

cultivated in LB media containing ampicillin at 37℃ with vigorous shaking at 200

123

rpm for overnight. 1% of the fresh culture was then inoculated into 20 mL of fresh

124

LB media containing ampicillin and cultivated at 37℃ with vigorous shaking at 200

125

rpm. When OD600 reached 0.5, isopropyl-β-d-thiogalactoside (IPTG) was added into

126

the culture at the final concentration of 0.5 mM. The culture was then cultivated at

127

20℃ with vigorous shaking at 200 rpm for 7 h. Cell extracts were prepared by

128

sonication and cell debris were removed by centrifugation as described previously.

129

The protein samples were then prepared for SDS-PAGE analysis in a 12%

130

polyacrylamide gel 14. The gel was scanned by BioSpectrum Imaging System (USA).

131

The content of the recombinant protein (CRP) was calculated according to O.D.

132

value 15 analyzed by BioSpectrum Imaging System software. 7



133 134

Optimization of induced expression of the recombinant ARO8 in E. coli

135

In order to obtain high-level expression of ARO8 in E.coli, culture conditions

136

including incubation temperature (16℃, 20℃, 25℃, 30℃, and 35℃), incubation

137

time (3 h, 5 h, 7 h, 9 h, and 11 h), and IPTG concentration (0 mM, 0.2 mM, 0.4 mM,

138

0.6 mM, 0.8 mM, and 1.0 mM) were firstly selected by the single-factor test 16. The

139

effects of three significant factors on protein expression were further evaluated using

140

the orthogonal experimental design L9 (33) 17, resulting in the optimal conditions for

141

expression of the recombinant ARO8.

142 143

Purification of the recombinant ARO8

144

The clear supernatant of cell debris were firstly filtered through a 0.22 µm pore

145

membrane. The filtered supernatant was then added to Ni column containing

146

Ni-NTA Resin (TRANSGEN, Beijing, China). Maltose was added to suppress

147

nonspecific binding 18. The column was washed with lysis buffer to remove unbound

148

proteins. The recombinant ARO8 attached to Ni2+-chelated beads in the Ni column

149

via the 6×His tag was eluted from the column using the washing buffer (50 mM

150

potassium phosphate solution, 2 mM EDTA, 2 mM DTT, 0.1 mM PLP, 200 mM

151

imidazole, pH 7.5). The eluate was collected for SDS-PAGE analysis 15, 19, 20.

152 153

Methionine production catalyzed by purified recombinant ARO8

154

100 µL of purified recombinant ARO8, 700 µL of lysis buffer, 100 µL of 10 mM 8


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KMBA, and 100 µL of 10 mM amino donor were mixed and incubated at 30℃ for

156

20 min. The enzymatic reaction solution was then diluted 10 times and used for

157

reversed phase HPLC (SHIMADZU, Shanghai, China) determination of D- and

158

L-methionine. HPLC analysis conditions were as follows: Inertsil ODS-SP column

159

(4.6×250 mm, 5 µm, SHIMADZU, Shanghai, China); injection volume 20 µL; oven

160

temperature 35℃; flow rate 0.5 mL/min; the eluate was monitored at 254 nm.

161

Mobile phase A (2 mM sodium acetate buffer containing 1 mM CuSO4, pH 4.0) and

162

phase B (acetonitrile) ran on an isocratic elution program (95% phase A buffer, 5%

163

phase B buffer).

164

The optimal amino donor was screened from 19 different amino acids by adding

165

them into the enzymatic reaction system, respectively, and evaluate their effects on

166

methionine production. The amount of methionine was determined by HPLC as

167

described previously and one unit of aminotransferase activity was defined as

168

formation of 1 µM methionine in 20 min. The molar ratio of the optimal amino

169

donor to KMBA was further optimized to improve the yield of methionine.

170 171

RESULTS AND DISCUSSION

172 173

Methionine biosynthesis in the S. cerevisiae strain with ARO8 expression

174

The wild type strain S288c, the recombinant strains S0 harboring the empty vector

175

and S8 harboring the ARO8 expression vector pYES-pgk-ARO8 were cultivated for

176

24 h to determine the aminotransferase activity for methionine biosynthesis. As 9



177

shown in Fig. 2, S8 exhibited the strongest aminotransferase activity, which was

178

more than 2-fold higher compared to S0 and S288c, indicating that expression of

179

ARO8 significantly increased the enzymatic activity and correspondingly enhanced

180

the generation of methionine using the substrate KMBA. As our previous work

181

proved that ARO8 played an effective role in methionine transamination 9, it

182

revealed that the aminotransferase ARO8 not only transformed methionine into the

183

corresponding α-keto acids via the Ehrlich pathway 9, but also reversibly acted on

184

KMBA to generate methionine when glutamate served as the amino donor.

185

Obviously, the transamination reaction between amino acids and α-keto acids

186

catalyzed by ARO8 was reversible. Karsten et al.

187

α-aminoadipate

188

dicarboxylic acid substrates such as L-tyrosine, L-phenylalanine, α-ketoadipate, and

189

L-α-aminoadipate. And it’s demonstrated that ARO8 could transform α-ketoglutarate,

190

α-ketoadipate, α-aminoadipate, phenylpyruvate, and 2-oxoglutarate into the

191

corresponding amino acids

192

seem to have been well characterized and investigated, this work gives evidences for

193

the reversible catalysis of ARO8 in transformation between L-methionine and

194

KMBA for the first time, contributing to comprehensive understanding of the

195

important aminotransferase ARO8 from yeast.

aminotransferase, which could

21

utilize both aromatic and

10, 21, 22

. Though ARO8 and its broad substrates range

196 197

categorized ARO8 as a

Induced expression of recombinant ARO8 in E. coli

10


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Sequence analysis showed that the 1503-bp ARO8 amplified from S. cerevisiae

199

S288c shared a 100% identity with the 2-aminoadipate transaminase gene (GenBank

200

accession no. NM_001181067.1). The recombinant expression vector pEASY-ARO8

201

constructed by inserting ARO8 into pEASY Blunt E1 was transformed into E. coli

202

BL21 (DE3), generating the recombinant strain BL21-ARO8. The protein samples of

203

BL21-ARO8 and BL21 cultivated with and without IPTG induction were analyzed

204

by SDS-PAGE. As shown in Fig. 3, a large number of the recombinant ARO8

205

proteins with the molecular mass of 57 kDa were observed in the supernatant of

206

ARO8-BL21 lysate with IPTG induction (Lane 1). In sharp contrast, the

207

recombinant ARO8 was almost hardly detected in the precipitate of ARO8-BL21

208

lysate with IPTG induction (Lane 2), the empty control strain BL-21 lysate with

209

IPTG induction (Lane 5, 6), and all the strains lysate without induction (Lane 3, 4, 7,

210

8). Furthermore, aminotransferase activity assay indicated that the supernatant of

211

BL21-ARO8 with IPTG induction exhibited the strongest activity, which was more

212

than 2-fold higher than that of all the strains without induction (Fig. 4). Heterologous

213

expression of eukaryotic genes in E. coli commonly generates inclusion body

214

formation and proteolytic degradation issues

215

recombinant protein without activity. However, in this work, the recombinant ARO8

216

was almost all expressed in the soluble form and showed a strong activity for

217

methionine biosynthesis, revealing that the recombinant ARO8 was efficiently

218

expressed and correctly folded in E. coli BL21. And obviously it’s much more

23

, and consequently leads to the

11



219

convenient to purify the soluble recombinant ARO8 from the supernatant of cell

220

lysate.

221 222

Optimization of induced expression and purification of recombinant ARO8 in E.

223

coli

224

In order to obtain a high yield of ARO8, the single-factor test and orthogonal

225

experimental design were performed to optimize the induced protein expression.

226

According to the single-factor test results (Fig. S1-S3), incubation temperature (20℃,

227

25℃, and 30℃), incubation time (5 h, 7 h, and 9 h), and IPTG concentration (0.5

228

mM, 0.6 mM, and 0.7 mM) were selected as the significant effect factors and further

229

evaluated using the orthogonal experimental design L9 (33) (Table 2 & 3).

230

Expression of ARO8 under 9 combined conditions were quantitatively analyzed and

231

shown in Table 3. Based on the range analysis result, the optimal condition for

232

protein expression was determined as the combination of A2B2C3, under which the

233

content of the recombinant protein (CRP) ARO8 reached 15.81%. Consequently, the

234

recombinant ARO8 was prepared by cell culture at 25℃ for 9 h with 0.7 mM IPTG

235

induction.

236

The recombinant ARO8 was eluted from the Ni column with the washing buffer

237

containing 200 mM imidazole and analyzed by SDS-PAGE. The electrophoresis

238

result showed that the highly concentrated 57 kDa ARO8 was detected in the eluted

239

sample and nonspecific protein was hardly observed in the same lane (Fig. 5). The

240

protein content measurement indicated that the concentration of ARO8 in the eluted 12


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sample reached 6.16 g/L, which was 6-fold higher than that in the sample without

242

purification, demonstrating the recombinant ARO8 was successfully purified. The

243

purified ARO8 was then used for methionine production.

244 245

Methionine production catalyzed by purified recombinant ARO8

246

The purified ARO8, KMBA, and amino donor were mixed and incubated for

247

methionine production assay. HPLC analysis results showed that the isomers D- and

248

L-methionine in the standard sample were detected and successfully separated, the

249

retention times of which were 11.9 min and 13.0 min (Fig. 6A) , respectively. In

250

sharp contrast, only L-methionine was detected in the sample of the reaction solution

251

catalyzed by ARO8 (Fig. 6B), indicating the distinct advantage of enzymatic

252

catalysis in L-methionine production.

253

Nineteen proteinogenic amino acids were selected as the amino donors to test their

254

influence on L-methionine production catalyzed by ARO8 using KMBA as the

255

amino receptor. A significant difference was observed among 19 amino acids. As is

256

shown in Fig. 7, higher yields of L-methionine were achieved when leucine,

257

glutamate, tryptophan, phenylalanine, aspartate, and tyrosine were used as amino

258

donor, respectively, demonstrating the broad-substrate transaminase activity of

259

ARO8 7. But other amino acids such as threonine, proline, glycine, serine, lysine,

260

and arginine as amino donor hardly resulted in methionine production, revealing the

261

substrate selectivity of ARO8. Considering the availability and market price, the

262

industrial bulk chemical glutamate was chosen as the optimal amino donor for 13



263

L-methionine production. Furthermore, the molar ratio of glutamate to KMBA was

264

further optimized for higher L-methionine yield. It’s indicated that more

265

L-methionine was produced as the molar ratio of glutamate to KMBA increased (Fig.

266

8). When the molar ratio of glutamate to KMBA reached 3:1, 0.99 mM L-methionine

267

was detected in the reaction system using 1 mM KMBA as the substrate. And

268

approximatively 100% KMBA was transformed into L-methionine when the molar

269

ratio of glutamate to KMBA was higher than 3:1 (Fig. 8). Consequently, an

270

approximatively 100% conversion rate of KMBA into L-methionine was achieved

271

under the optimal condition that a 3:1 molar ratio of glutamate / KMBA were

272

catalyzed by ARO8.

273

Continuous attempts have been made to produce biologically active L-methionine

274

by enzymatic catalysis. Tokuyama and Hatano

275

DL-methionine could be converted into L-methionine by catalysis of N-acylamino

276

acid racemase and L-aminoacylase with a conversion rate of 90%. Though the

277

conversion rate is not extremely high, the price advantage of the substrate N-acetyl

278

DL-methionine makes it easier to fulfill the industrial-operated process for

279

L-methionine production. Ishikawa et al. 25 reported a remarkable conversion rate of

280

98% from DL-5-(2-methylthioethyl)-hydantoin into L-methionine. However, the

281

high market price of the substrate DL-5-(2-methylthioethyl)-hydantoin and the

282

complex reaction process catalyzed by multiple enzymes including hydantoin

283

racemase, hydantoinase, and N-carbamyl-L-amino acid amidohydrolase bring

284

challenges for industrialized production. In contrast, the L-methionine production

24

14


reported that N-acetyl

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methods using KMBA and glutamate by ARO8 catalysis reported in this work has its

286

unique strength that it is a highly efficient production method without complex

287

reaction process. And this is an asymmetric synthesis mode to produce the

288

L-methionine, as one reaction mode for transaminases.

289

In conclusion, the aminotransferase ARO8 involved in fusel alcohol

290

biosynthesis via the Ehrlich pathway was cloned from S. cerevisiae and

291

over-expressed in E. coli. The recombinant 57 kDa ARO8 protein was successfully

292

purifed and its reversible transamination activity for conversion of KMBA into

293

L-methionine was confirmed by enzymatic catalysis assay in vitro for the first time.

294

The conversion rate of KMBA into L-methionine reached approximately 100% by

295

screening the amino donor and optimizing the molar ratio of the amino donor and

296

receptor, providing a new highly efficient method for L-methionine production. The

297

substrate KMBA for L-methionine production used in this work is not easily

298

accessible, which seems to be a barrier for industrial process. However, van der

299

Heijden et al. 26 developed an efficient one-pot oxidation/Passerini/hydrolysis

300

sequence procedure for the formal α-carboxylation of primary alcohols, by which

301

methional possibly could be converted into KMBA by enzymatic catalysis or

302

chemical synthesis, providing the precursor for L-methionine biosynthesis by

303

transamination. Consequently, it might be reasonable to propose a possible novel

304

route for industrial production of L-methionine using methional as the substrate by a

305

combination of chemical synthesis and enzymatic catalysis.

306 15



307 308

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AUTHOR INFORMATION ⊥

Yiping Wu and Musu Zha contribute to this work equally.

309

*Corresponding Author. Phone: 86-10-68985252.

310

[email protected] (Sheng Yin); [email protected] (Chengtao Wang)

Fax: 86-10-68985252.

Email:

311 312

FUNDING SOURCES

313

This work was supported by National Natural Science Foundation of China (NSFC

314

31401669 & 31571801), Beijing Natural Science Foundation (5154027), National

315

Key Research and Development Program of China (2016YFD0400802), Beijing

316

Municipal Science and Technology Project (Z171100002217019), Support Project of

317

High-level Teachers in Beijing Municipal Universities (IDHT20180506), and

318

Science

319

(PXM2018-014213-000033).

and

Technology

Achievement

Transformation

Upgrade

Project

320 321

SUPPORTING INFORMATION

322

Figure S1. Effect of different incubation temperature on the content of the

323

recombinant ARO8 in the strain BL21-ARO8

324

Figure S2. Effect of different incubation time on the content of the recombinant

325

ARO8 in the strain BL21-ARO8

326

Figure S3. Effect of different IPTG concentrations on the content of the recombinant

327

ARO8 in the strain BL21-ARO8

328 16


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3. Obata, F.; Miura, M. Enhancing S-adenosyl-methionine catabolism extends

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4. Willke, T. Methionine production—a critical review. Appl. Microbiol. Biotechnol.

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engineering of Corynebacterium glutamicum for methionine production by removing

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feedback inhibition and increasing NADPH level. Antonie van Leeuwenhoek. 2016,

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6. Kumar, D.; Gomes, J. Methionine production by fermentation. Biotechnol. Adv.

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Figure captions

Figure 1. The metabolism of methionine via the Ehrlich pathway in S. cerevisiae (black arrow) and the route for methionine production proposed in this work (red arrow)

Figure 2. Aminotransferase activity assay in S. cerevisiae strains

Figure 3. SDS-PAGE analysis of protein samples from E. coli strains M, protein marker; Lane 1, supernatant of ARO8-BL21 lysate with IPTG induction; Lane 2, precipitate of ARO8-BL21 lysate with IPTG induction; Lane 3, supernatant of ARO8-BL21 lysate without induction; Lane 4, precipitate of ARO8-BL21 lysate without induction; Lane 5, supernatant of BL21 lysate with IPTG induction; Lane 6, precipitate of BL21 lysate with IPTG induction; Lane 7, supernatant of BL21 lysate without induction; Lane 8, precipitate of BL21 lysate without induction; recombinant ARO8 was indicated by red arrow.

Figure 4. Aminotransferase activity assay in E. coli strains

Figure 5. SDS-PAGE analysis of purified recombinant ARO8 M, protein marker; Lane 1, the protein sample containing ARO8 without purification; 21



Lane 2, the protein sample of purified ARO8

Figure 6. Reversed phase HPLC analysis of the mixed standard sample of Dand L-methionine (A) and the sample of methionine catalyzed by purified recombinant ARO8 (B)

Figure 7. Effect of different amino acid donors on L-methionine production catalyzed by purified recombinant ARO8

Figure 8. Effect of different molar ratios of KMBA to glutamate on L-methionine production catalyzed by purified recombinant ARO8

22


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Page 23 of 29


Tables

Table 1 Strains and plasmids used in this work Strains or plasmids

Relevant features

Reference or source

Vector for protein expression, Amp R

TRANSGEN Inc., Beijing, China

Plasmids

pEASY-Blunt E1 Strains

E. coli Trans-T1

Host for cloning

TRANSGEN Inc., Beijing, China

E. coli BL21 (DE3)

Host for protein expression

TIANGEN Inc., Beijing, China

MATα SUC2 gal2 mal mel flo1 flo8-1 hap1 ho bio1 bio6; donor of ARO8

Laboratory collection

S. cerevisiae S288c harboring empty expression vector pYES-pgk, G418 R

Yin S et al., 2014

S. cerevisiae S288c (ATCC 204508)

S. cerevisiae S0

S. cerevisiae S288c harboring pYES-pgk-ARO8, G418 R

S. cerevisiae S8

Yin S et al., 2014

Table 2 Factors and levels for L9 (33) orthogonal experimental design Levels

Factors A (℃)

a

B (h) b

C (mM) c

1

20

7

0.5

2

25

9

0.6

3

30

11

0.7

a, incubation temperature; b, incubation time; c, IPTG concentration.

23



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Table 3 The design matrix and experimental data for optimizing induced expression of ARO8 Run A 1 2 3 4 5 6 7 8 9 K1 a K2 a K3 a Rb Qc

CRP（（%））

Factors d

1 1 1 2 2 2 3 3 3 11.21 14.47 7.96 6.52 A2

B

e

1 2 3 1 2 3 1 2 3 10.71 11.78 11.15 1.07 B2

C

f

1 2 3 2 3 1 3 1 2 10.01 11.25 12.38 2.38 C3

9.10 10.94 13.60 15.29 15.81 12.32 7.74 8.60 7.53

a, the average value of each level; b, the range value for each factor; c, the best group; d, incubation temperature; e, incubation time; f, IPTG concentration.

24


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Figures

Aminotransferase activity (U/mg)

Figure 1

1.8 1.5 1.2 0.9 0.6 0.3 0 S8

S0 S288c S. cerevisiae strains

Figure 2 25



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Figure 3


15 12 9 6 3 0 ARO8-BL21(induction)

ARO8-BL21 E.coli strains

Figure 4

26


BL21

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Figure 5

Figure 6

27




120 100

80 60

40

20 0 Lys Trp Phe Leu Ile Thr Val Asn Gly Ala Pro Ser Tyr Cys Gln His Asp Glu Arg Amino acid

Figure 7

Figure 8

Graphic for table of contents 28


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A novel method for L-methionine production catalyzed by the

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