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Generation of 2-furfurylthiol by carbon-sulfur lyase from Baijiu yeast Saccharomyces cerevisiae G20 Musu ZHA, Baoguo Sun, Sheng Yin, Arshad Mehmood, Lei Cheng, and Chengtao Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06125 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 13, 2018

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Page 1 of 34

Journal of Agricultural and Food Chemistry

1

Generation of 2-furfurylthiol by carbon-sulfur lyase from Baijiu yeast

2

Saccharomyces cerevisiae G20

3 4

Musu Zha†‡, Baoguo Sun†‡, Sheng Yin*†‡, Arshad Mehmood†‡, Lei Cheng†‡, Chengtao

5

Wang*†‡

6 7

†

8

Technology & Business University, Beijing 100048, China.

9

‡

10

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

Beijing Engineering and Technology Research Center of Food Additives, Beijing

Technology & Business University, Beijing 100048, China

11 12

*Corresponding Author

13

Phone: 86-10-13641119958. Fax: 86-10-68985252. E-mail: [email protected].

14


ACS Paragon Plus Environment


15

ABSTRACT:

16

2-Furfurylthiol is the representative aroma compound of Chinese sesame-flavored

17

Baijiu. Previous studies demonstrated that Baijiu yeasts could generate 2-furfurylthiol

18

using furfural and L-cysteine as precursors, and the Saccharomyces cerevisiae genes,

19

STR3 and CYS3, are closely related to 2-furfurylthiol biosynthesis. To confirm the

20

mechanism of STR3 and CYS3 gene products on 2-furfurylthiol biosynthesis, their

21

encoded proteins were purified and we confirmed their activity as carbon-sulfur lyase.

22

Str3p and Cys3p were able to cleave the cysteine-furfural conjugate to release

23

2-furfurylthiol. Moreover, the characterization of the enzymatic properties of the

24

purified proteins showed good thermal stability and wide pH tolerance levels, which

25

enable their strong potential for various applications. These data provide direct

26

evidence that yeast Str3p and Cys3p release 2-furfurylthiol in vitro, which can be

27

applied to improve Baijiu flavor.

28

KEY WORDS: 2-furfurylthiol, Chinese sesame-flavored Baijiu, Saccharomyces

29

cerevisiae, carbon-sulfur lyase, Str3p, Cys3p, cysteine-furfural conjugate


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INTRODUCTION

31

2-Furfurylthiol is a typical potent odorant in Chinese sesame-flavored Baijiu.1

32

Baijiu yeasts produce this compound by the utilization of furfural and L-cysteine as

33

precursors.2 It has also been reported that cysteine-S-conjugated compounds are the

34

precursors of 2-furfurylthiol and other aromatic thiols (3-mercaptohexan-1-ol [3MH]

35

and 4-mercapto-4-methylpentan-2-one [4MMP]), which contribute to aroma

36

descriptors such as roasted sesame, grapefruit, passion fruit and boxwood in many

37

fermented beverages, with a sensory perception threshold range in parts per trillion.3-9

38

It is generally accepted that 2-furfurylthiol and other aromatic thiols are produced

39

during the biotransformation of cysteine-S-conjugate by various bacteria possessing

40

carbon-sulfur (C-S) β-lyase enzymatic activity.8,

41

microorganisms have been isolated and studied for their ability to catalyze

42

cysteine-S-conjugates. Many species, including Escherichia coli, Eubacterium

43

limosum, Staphylococcus haemolyticus, Streptococcus anginosus, have been reported

44

to possess C-S β-lyase activity and contribute to the release of volatile thiol;12-15

45

however, most of them are not food-grade. Few food-grade microorganisms possess

46

(C-S) β-lyase activity.13 It is worth noting that the yeast cystathionine β(γ)-lyase was

47

reported to enable the release of volatile thiols from cysteine-conjugated precursors

48

by cleaving a C-S bond.16, 17 Apart from their potential role in aromatic thiol release,

49

cystathionine β-lyase (CBL; EC 4.4.1.8) and cystathionine γ-lyase (CGL; EC 4.4.1.1)

50

are involved in the biosynthesis of methionine and cysteine.17 CBLs catalyzes the

10,


11

To date, numerous


51

conversion of L-cystathionine into L-homocysteine, while CGLs transforms

52

cystathionine into L-cysteine.18 Although their main physiological role is different in

53

conversion of cystathionine, they can also possess C-S β-lyase activity and help

54

release the odorous volatile thiol.19

55

Several yeast genes (IRC7, STR3, CYS3, BNA3 and GLO1) have been reported to

56

be involved in volatile thiol release.17, 19-21 Among them, the IRC7 and STR3 genes

57

have been confirmed to be responsible for 4MMP and 3MH production due to their

58

encoded carbon-sulfur β-lyase activity.3, 22 Moreover, the S. cerevisiae genes, STR3

59

and CYS3, have been recently found to encode cystathionine β(γ)-lyase, which could

60

be closely related to 2-furfurylthiol production during Chinese sesame flavored Baijiu

61

fermentation.2 However, there is no direct evidence to suggest that yeast cystathionine

62

β(γ)-lyase could cleave the C-S bond of the cysteine-furfural conjugate and release

63

2-furfurylthiol.

64

Therefore, the aims of the present study were (i) to purify the S. cerevisiae gene

65

products, Str3p and Cys3p, and confirm their β-(C-S) lyase activity, and (ii) to provide

66

direct evidence that a purified form of the yeast enzymes has activity toward the

67

cysteine-S-conjugated precursors of 2-furfurylthiol.

68

Materials and Methods

69

Chemicals. 2-Furfurylthiol (98%), furfural (98%), L-cysteine (99%), L-cystathionine

70

(98%), dichloromethane (99.9%), K2HPO4, KOH, KCl, and glycerol were purchased

71

from Sigma-Aldrich (Beijing, China). The cysteine-furfural conjugate was prepared


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72

according to the method developed by Huynh-Ba et al.8

73

Microbial Strains, Medium, and Culture Conditions. Chemically competent E. coli

74

DH5α and BL21(DE3) cells (TIANGEN Inc., Beijing, China) were used for the

75

amplification of plasmid DNA and protein expression, respectively (Table 1). Growth

76

and selection were carried out in lysogeny broth (LB) medium supplemented with 200

77

µg/mL ampicillin. The Baijiu yeast, S. cerevisiae G20 was previously isolated from

78

samples of Daqu from Chinese sesame-flavored Baijiu. S. cerevisiae S288c (ATCC

79

204508) was used as the reference strain. The yeasts were inoculated into yeast

80

extract−peptone−dextrose (YPD) medium.

81

Fermentation Conditions. The fermentation medium was prepared according to the

82

previously reported method.2 Briefly, 500 g of milled sorghum powder was added to 2

83

L of deionized water, and the mixture was boiled for 2 h, followed by saccharification at

84

60 °C for 4 h with the addition of glucoamylase (5 U/L). Subsequently, the mixture was

85

filtered through gauze and centrifuged at 8000 g for 15 min. The supernatant was

86

collected and used as the sorghum-extract medium. The sugar content was measured on

87

a Leica refractometer (Fisher Scientific, Pittsburgh, PA, USA) and diluted with water to

88

a final sugar concentration of 10 °Bx (95±5 g/L reducing sugar), followed by

89

sterilization at 121 °C for 15 min before use. Moreover, 0.96 g/L (10 mM) furfural and

90

1.21 g/L (10 mM) L-cysteine solution were prepared as precursor compounds, were

91

purified by filtration, and were mixed with the sterilized sorghum-extract medium.

92

Another precursor cysteine-furfural conjugate (3, 6 and 10 mM) was also prepared



Page 6 of 34

93

similarly and added to the sterilized extract medium. S. cerevisiae S288c and G20 were

94

pre-cultured, respectively, in YPD liquid medium at 30 °C, for 24 h. Subsequently, 2%

95

(v/v) of the inoculum was inoculated into 250 mL Erlenmeyer flasks containing 100 mL

96

of sterile liquid fermentation medium. Stationary fermentations were conducted at

97

30 °C for 48 h.

98

Quantitative Real-Time RT-PCR. Total RNA was prepared according to the protocol

99

developed by Chen et al.23 with minor modifications. The total RNA was extracted

100

using the RNA Prep Micro Sample RNA extraction kit (Bio Teke, Beijing, China)

101

according to the manufacturer’s instructions, and its quality was evaluated using

102

NanoDrop assay (Thermo Fisher Scientific, San Jose, CA, USA). Subsequently, cDNA

103

was synthesized from the total RNA using Superscript III reverse transcriptase

104

(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

105

The obtained cDNA samples were used for quantitative real-time PCR (RT-qPCR),

106

which was performed using a Bio-Rad CFX96 real-time detection system with

107

SuperReal PreMix (SYBR Green) reagent (Tiangen, China). To quantitate the

108

transcript level of the CYS3, and STR3 genes, the data were normalized using ACT1 as

109

the reference transcript. The primer pairs used were as follows: CYS3 (CYS3.F,

110

5′-TTGACGACTTGGTTAGAATC-3′; CYS3.R, 5′-TTAGTTGGTGGCTTGTTTC-3′),

111

STR3

112

5′-CGTCACAGCCCATATACTCAG-3′),

113

5′-GCCAAGATAGAACCACCAATCC-3′;

(STR3.F,

5′-TCAAACCTACCAGAACAAACAAG-3′; and


ACT1

STR3.R, (ACT1.F, ACT1.R,

Page 7 of 34


114

5′-CTGATGTCGATGTCCGTAAGG-3′). The threshold cycle (CT) values were

115

determined from duplicate fermentations, and gene expression was normalized against

116

the ACT1 reference gene by the 2−∆∆CT method.

117

Vector Construction. The strains and plasmids used in this work are presented in Table

118

1.Vector construction was performed according to a previously reported method with

119

minor modifications.2 The S. cerevisiae STR3 and CYS3 gene were amplified from

120

genomic

121

(5′-ATGCCGATCAAGAGATTAGATAC-3′),

122

(5′-TTACAATTTCGAACTCTTAATATTC-3′),

123

(5′-ATGACTCTACAAGAATCTGATAAAT-3′)

124

(5′-TTAGTTGGTGGCTTGTTTC-3′). The STR3 and CYS3 coding regions were

125

cloned and inserted into the multiple cloning sites (MCS) of the vector pEASY-E1

126

(Transgen, Beijing, China), respectively. The resultant recombinant vectors were

127

transferred into E. coli DH5α. The transformants were selected on LB agar plates

128

supplemented with ampicillin (200 µg/mL), and the corresponding recombinant strains

129

were further confirmed by PCR and sequencing. The final Str3p and Cys3p were

130

expressed in E. coli BL21 (DE3).

131

Protein Expression and Purification. According to a previous study,3 the

132

transformants were grown in 100 mL of LB medium to the log phase (optical density at

133

600 nm [OD600] of 0.4 to 0.6) from an overnight culture supplemented with 200 µg/mL

134

ampicillin. The expression of recombinant protein was induced with 0.5 mM

DNA from

the

Baijiu

yeast

strain

G20


with

primers

str3-f str3-r cys3-f

and

cys3-r


Page 8 of 34

135

isopropyl-β-D-thiogalactopyranoside (IPTG) at 20 °C. After 10 h, bacterial cells were

136

harvested by centrifugation and washed twice with 50 mM phosphate buffer (pH 7.0).

137

The resulting cell pellets were resuspended in cell lysis buffer (50 mM Tris, pH 8.0, 100

138

mM KCl, 10% [wt/vol] glycerol, 0.25 mM Triton X-100, 10 mM imidazole, 100 µM

139

PLP) and lysed by ultrasonication. The lysates were centrifuged at 10625 g for 10 min

140

and filtered through a 0.22-µm-pore-size (Millipore) filter. The resulting cleared lysates

141

were purified by using ProteinIso™ Ni−NTA Resin (TransGen Biotech, China)

142

according to the protocol with NiA buffer containing 20 mM Tris, pH 7.0, 250 mM KCl,

143

100 µM PLP, and 200 mM imidazole. The purified enzymes were adjudged

144

homogeneous after examination by SDS-PAGE. Protein concentrations were

145

determined by the Coomassie brilliant blue dyeing method, using bovine serum

146

albumin (BSA) as the standard.

147

Lyase Activity Assay and Characterization of Enzymatic Properties. Reactions

148

were carried out according to a previous study with minor modifications.3 The

149

conditions of the reactions (0.5 mL) were as follows: 20 µg/mL of protein, 50 mM

150

phosphate buffer, pH 7.0, 20 µM PLP, 1 mM EDTA, and 5 mM L-cystathionine. The

151

reaction mixtures were incubated for 20 min at 40 °C and then stopped by boiling for 2

152

min.

153

2,4-dinitrophenylhydrazine and 5 mL 0.4 M NaOH, and then stewed for 30 min at room

154

temperature. The absorbance was measured at 520 nm. For optimum pH tests, the

155

reaction buffers were titrated at a pH ranging from 5 to 11 (pH 5.0−8.0, PBS; pH

The

mixtures

were

cooled

and

reacted


with

0.5

mL

1

mM

Page 9 of 34


156

8.0−11.0, Tris-HCl buffer). To determine the optimal temperature, the reactions were

157

conducted at pH 7.0 for 30 min at a temperature ranging from 20 to 70 °C. The relative

158

activity was measured as described above.

159

To investigate the thermal stability and pH stability of the enzyme, the enzyme was

160

kept at different temperatures in reaction buffers at pH 7.0 or at different pH values at

161

40 °C for 2 h, and then L-cystathionine was put into the reaction system as a substrate to

162

measure the relative activity

163

All experiments were performed in triplicate, and enzyme-free controls were

164

included. One unit of enzyme activity was defined as the amount of enzyme required to

165

produce 1 µmol of ketonic acid per min at 40 °C and pH 7.0.

166

Enzymatic Reactions with Cysteine-Furfural Conjugate. The reactions were as

167

follows. Cleavage of the cysteine-furfural conjugate was performed in a total volume of

168

1 mL. For the negative controls, reactions were performed with Str3p and Cys3p that

169

were heat inactivated for 2 min at 95 °C and the extract from induced E. coli cells

170

transformed with an empty pEASY-E1 vector. The reaction buffers were as follows: 50

171

µg/mL enzyme, 50 mM phosphate buffer, pH 7.0 or 8.0, 20 µM PLP, and 1 mM EDTA

172

with 3 or 6 mM substrate, incubated at 30 °C for 1 h and kept at 4 °C until assayed. An

173

aliquot of 500 µL of the enzymatic reaction mixtures was extracted 2 times with

174

dichloromethane (GC grade), and the extracts were centrifuged at 10625 g for 5 min at

175

4 °C. The organic phase was collected, filtered through a 0.22-µm-pore-size (Millipore)

176

filter, and stored at −20 °C before GC−MS/MS analysis. All experiments were



177

performed in triplicate.

178

Gas Chromatography−Mass Spectrometry Analysis. The GC−MS analysis was

179

performed on a Thermo Scientific TRACE 1310 GC system coupled to a TSQ8000

180

EVO mass selective detector (Thermo Fisher Scientific) and a TG-5MS column (30 m

181

× 0.25 mm i.d., 0.25-µm film thickness; Thermo Fisher Scientific). The instrument

182

conditions were as described in a previous study reported by Zha et al.2 with the minor

183

modifications. The fermentation sample was pretreated and measured according to the

184

method of Zha et al.2 In addition, quantitation of 2-furfurylthiol in the enzymatic

185

reaction mixture was carried out by GC-MS/MS in selected reaction monitoring (SRM)

186

mode (MS/MS, retention times), and were recorded as follows: furfural (96/95, 5.29

187

min), 2-furfurylthiol (81/53, 6.52 min). The compounds were clearly identified by

188

comparison to reference spectra (NIST MS Search 2.0) and pure standards.

189

RESULTS AND DISCUSSION

190

The expression of STR3 and CYS3 genes in S. cerevisiae. Zha et al.2 demonstrated

191

that S. cerevisiae genes, STR3 and CYS3, were closely related to 2-furfurylthiol

192

production. Thus, we investigated the expression of STR3 and CYS3 genes at different

193

phases (6 and 12 h) of S. cerevisiae S288c and G20 fermented with/without substrate

194

(10 mM furfural and L-cysteine). The expression levels of both the STR3 and CYS3

195

genes were increased in both strains when furfural and L-cysteine were added in the

196

medium. Notably, in G20, the STR3 and CYS3 transcript levels were 2.89- and

197

2.08-fold higher than those in fermentation without added substrate at 6 h, respectively


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198

(Figure 1C, D). However, in S288c, the relative expression increase of STR3 and CYS3

199

was < 1.5-fold (Figure 1A, B). The increased transcriptional activities of these two

200

genes indicated an increase in 2-furfurylthiol biosynthetic activity, which is in

201

accordance with previous findings.2 To confirm the relationship between the

202

expression of these genes and 2-furfurylthiol production, we cultured S. cerevisiae

203

G20 (high 2-furfurylthiol-producing strain) and S288c (reference strain) in

204

fermentation medium with substrate. The expression levels of the STR3 and CYS3

205

genes at different phases of S288c and G20 growth were analyzed by quantitative

206

real-time PCR (Figure 2). The expression levels of STR3 and CYS3 genes in G20 were

207

much higher than those in S288c at 3, 6, and 9 h, which could be the reason that G20

208

produced more 2-furfurylthiol than S288c.2 However, the expression patterns of the

209

STR3 and CYS3 genes in G20 were different from each other. STR3 transcript levels

210

(Figure 2A) decreased gradually, which were 15.24-, 9.55-, and 1.87-fold higher than in

211

the S288c at 3, 6, and 9 h, respectively. Interestingly, CYS3 transcript levels (Figure 2B)

212

gradually increased at 3 and 6 h (6.18- and 9.9-fold, respectively), but decreased during

213

the rest of fermentation process. STR3 and CYS3 levels showed strong positive

214

correlations with 2-furfurylthiol production compared with the observed expression

215

profiles and previously reported 2-furfurylthiol production.2

216

Effect of Substrates on 2-Furfurylthiol Biosynthesis. Our previous study

217

demonstrated that Baijiu yeasts could generate 2-furfurylthiol using furfural and

218

L-cysteine as precursors.

2

It has also been reported that 2-furfurylthiol biosynthesis was



219

achieved by coupling cysteine to furfural and cleaving the cysteine−furfural conjugate

220

into 2-furfurylthiol, pyruvic acid, and ammonia.8 Thus, the obtained cysteine-furfural

221

conjugate was investigated for the biogeneration of 2-furfurylthiol. As shown in

222

Figure 3, both S. cerevisiae S288c (Figure 3A) and G20 (Figure 3B) could release

223

0.55 and 6.84 mg/L 2-furfurylthiol from 10 mM cysteine-furfural conjugate,

224

respectively, which was 41 and 121% higher compared with the addition of 10 mM

225

furfural and L-cysteine. Moreover, S. cerevisiae G20 showed a strong catalytic

226

efficiency toward cysteine-furfural conjugate, which could provide significant

227

potential for improving Baijiu flavor. Moreover, furfural, particularly furfuryl alcohol,

228

was identified in the fermentation medium by GC-MS (data not shown). Furfural,

229

possibly originating from hydrolysis of the cysteine-furfural conjugate, was

230

enzymatically reduced into 2-furfuryl alcohol.2, 8 These observations confirmed that S.

231

cerevisiae S288c and G20 could catalyze the transformation of the cysteine-furfural

232

conjugate into 2-furfurylthiol, which corroborated the results of previous studies.8, 10

233

Nevertheless, little is known about the genetic regulation of 2-furfurylthiol synthesis,

234

and there is no direct evidence to suggest that yeast cystathionine β(γ)-lyase could

235

cleave the C-S bond of the cysteine-furfural conjugate and release 2-furfurylthiol.

236

Purification of S. cerevisiae Str3p and Cys3p. To verify the function of S. cerevisiae

237

STR3 and CYS3 gene products, we expressed recombinant Str3p and Cys3p in E. coli

238

and purified the protein by using Ni-NTA chromatography in the presence of its

239

cofactor (PLP). The analysis of the purified Str3p and Cys3p by SDS-PAGE is shown


Page 12 of 34

Page 13 of 34


240

in Figure 4. The monomeric recombinant Str3p (Figure 4A) and Cys3p (Figure 4B)

241

have the predicted molecular mass of 53 kDa and 42.5 kDa,3 and migrated at

242

approximately 52 kDa and 42 kDa on the SDS-PAGE gel, respectively. A yield of 50.4

243

mg and 72 mg pure proteins per liter of culture was typically obtained, respectively.

244

Holt et al.3 also purified the S. cerevisiae Str3p with a molecular size of 52 kDa, and

245

confirmed its ability to release aromatic thiols in vitro. Thus, we investigated the

246

characteristics of Str3p and Cys3p, and their influence on 2-furfurylthiol release.

247

Effect of pH and pH Stability on Enzyme Activity. The effect of pH and pH stability

248

on the activity of recombinant Str3p and Cys3p was investigated using the

249

physiological substrate, L-cystathionine. The enzyme activity was measured by the

250

detection of ketonic acid (pyruvate or α-ketobutyrate) at 520 nm. The Str3p exhibited a

251

good activity toward L-cystathionine at pH 7 to 9, and the optimum activity was at pH 8

252

(Figure 5A). At a pH between 6.0 and 10.0, the enzyme displayed a high relative

253

activity that was above 75%; however, the relative activity obviously decreased at pH 5

254

(23%). Moreover, the optimum activity of Cys3p was at pH 7 (Figure 5B), and there

255

was no significant activity below pH 6 or above pH 11 in both recombinant proteins. A

256

previous study demonstrated that recombinant Str3p display a bell-shaped pH-rate

257

profile and that the optimum activity was at pH 8.75,3 which is generally in agreement

258

with our findings. Both purified enzymes exhibited a relatively wide pH range between

259

pH 6 and 9, where they displayed more than 80% of the relative activity. These results

260

indicate that S. cerevisiae Str3p and Cys3p, purified from E. coli, are consistent with



261

cystathionine β(γ)-lyase from other microorganisms.24-26

262

Effect of Temperature and Thermal Stability on Enzyme Activity. The optimal

263

temperatures of the purified Str3p and Cys3p were 50 °C and 40 °C, respectively

264

(Figure 5). Str3p showed high relative activity at 40 and 60 °C with 98% and 94% of

265

the highest activity, respectively (Figure 5C). Notably, Cys3p showed high relative

266

activity (> 60%) from 30 to 60 °C (Figure 5D). The relative activity of both proteins

267

significantly decreased outside of the range from 30 to 60 °C. Both pure proteins were

268

stable below 40 °C, with >88% relative activity. However, the stability of the enzyme

269

decreased as the temperature increased, particularly when it exceeded 60 °C, where

270

only 5% of the initial activity was retained. The recombinant Str3p and Cys3p have

271

high optimal temperature and good thermal stability. The temperature optimum of the

272

purified enzyme activity was much higher than that reported for Lactobacillus

273

delbrueckii subsp. bulgaricus ATCC BAA-365 (25 °C).27 Generally, the thermostable

274

enzymes are often associated with stability and functionality during fermentation,

275

enabling the high potential for application of these enzymes in Chinese

276

sesame-flavored Baijiu and other fermented beverages.

277

Enzymatic Release of 2-Furfurylthiol from Cysteine-Furfural Conjugate

278

Precursor. Our previous study confirmed that S. cerevisiae genes, STR3 and CYS3,

279

contributed to 2-furfurylthiol biosynthesis;2 therefore, we speculated on the ability of

280

the encoded enzymes to release 2-furfurylthiol from the cysteinylated precursors.

281

GC-MS analysis of the extract of the enzymatic reaction mixture revealed one peak


Page 14 of 34

Page 15 of 34


282

responding to m/z 95 and 96, and another compound with m/z values of 53, 81, and 114.

283

The spectra were tentatively attributed to furfural at 5.29 min and 2-furfurylthiol at

284

6.52 min. Identification was confirmed by the injection of pure standard compounds,

285

indicating also that furfural and 2-furfurylthiol were actually present in the reaction

286

mixture described above. GC-MS/MS analysis of the reaction mixture exhibited two

287

peaks at 5.29 and 6.52 min for the transitions 96 → 95 and 81 → 53, respectively. The

288

analysis of pure standard compounds showed that these transitions and retention times

289

were characteristic of furfural and 2-furfurylthiol.

290

The in vitro reaction of purified Str3p and Cys3p with the cysteine-furfural conjugate

291

of 2-furfurylthiol confirmed our hypothesis that these enzymes have a residual

292

cysteine-S-conjugate-lyase activity and could cleave this substrate to release

293

2-furfurylthiol. As shown in Figure 6, S. cerevisiae Str3p was able to release 2.32 µM

294

2-furfurylthiol from 6 mM precursor when the reaction was conducted at pH 8, which

295

was 47.8% higher than that at pH 7 (Figure 6A). However, Cys3p preferred pH 7

296

solutions and produced 4.34 µM 2-furfurylthiol from 3 mM precursor, which was 75.7%

297

higher than that at pH 8 (Figure 6B). This special activity at pH 7 or 8 was consistent

298

with the pH rate profile of both enzymes with L-cystathionine as a substrate (Figure 5).

299

Interestingly, the production of 2-furfurylthiol was dependent on the concentration of

300

the precursor for both enzymes. The Cys3p displayed a preference for the

301

cysteine-furfural conjugate at a low concentration (3 mM), which was in contrast with

302

Str3p. Moreover, 2-furfurylthiol was not detected in the control groups (empty vector



303

control and heat-inactivated purified enzymes). Holt et al.3 had also found that Str3p

304

displayed a modest side activity toward the cysteinylated aroma precursors and

305

released the relevant thiols. Other studies confirmed the same cleavage reaction, carried

306

out by various bacteria possessing β-(C-S) lyase enzymatic activity for Cys-thiol

307

precursors.10, 13, 28, 29 It is generally accepted that the molar conversion rate of Cys-thiol

308

precursors to relevant thiols can range from 0.1% to 10%, with an average of less than

309

5%.21, 30-35 The conversion yields of 2-furfurylthiol in our study seemed to be higher in

310

vitro (4.34 µM/h) than in Baijiu fermentation (2.21 µM/h).2 Due to poor precursor

311

conversion rate and yeasts’ side enzyme activity toward Cys-thiol precursors, it could

312

be possible that the majority of precursors are degraded and used as nitrogen, sulfur and

313

carbon sources by yeast. This is consistent with our previous finding that a significant

314

amount of furfural (precursor) was transformed into furfuryl alcohol during

315

fermentation.2 Thus the untapped precursors in the fermentation medium are an

316

abundant source of aroma that could be revealed by developing more efficient

317

precursor-converting yeast strains.

318

To the best of our knowledge, this is the first direct evidence of purified yeast

319

enzymes (Str3p and Cys3p) displaying a CS β-lyase activity necessary to cleave the

320

cysteine-S-conjugates of 2-furfurylthiol (Figure 7). Moreover, the yeast Cys3p, a

321

cystathionine γ-lyase, was shown to cleave the cysteine-furfural conjugate and release

322

2-furfurylthiol for the first time. In conjunction with our previous fermentation

323

findings,2 we confirm that the enzymatic activity of Str3p and Cys3p is directly


Page 16 of 34

Page 17 of 34


324

responsible for 2-furfurylthiol production in Baijiu yeast. Moreover, the purified

325

enzymes have a good thermal stability and wide pH tolerance level, which enable their

326

strong potential for use in food and beverages.

327

ASSOCIATED CONTENT

328

Supporting Information

329

GC-MS analysis, comparison of GC-MS/MS chromatograms

330

AUTHOR INFORMATION

331

*Corresponding Author.

332


333


334

FUNDING

335

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

336

Grant No. 31571801 and 31401669), National Key Research and Development

337

Program (Grant No.2016YFD0400802 and 2016YFD0400502), Beijing Municipal

338

Science and Technology Project (Grant No. Z171100002217019) and the Construction

339

of the Scientific and Technological Innovation Service capacity of the Beijing

340

Municipal Education Committee(PXM2016-014213-000034).

341

NOTES

342

The authors declare no competing financial interest.



343

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452

Effect of Saccharomyces cerevisiae yeast strains on the liberation of volatile thiols in

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Sauvignon blanc wine. Am. J. Enol. Viticult. 2001, 52, 136-139.

454



455

Figure legends

456

Figure 1. Determination of STR3 and CYS3 gene expression levels in S. cerevisiae

457

S288c (A, B) and G20 (C, D) fermented with/without substrate (furfural and

458

L-cysteine);

459

experiments were repeated three times. Data are the average of three independent

460

experiments. Error bars represent ± SD.

ACT1 gene was used as the internal control in RT-qPCR, and the

461 462

Figure 2. Expression pattern of STR3 (A) and CYS3 (B) genes in S. cerevisiae G20

463

compared with S288c; furfural and L-cysteine were used as substrates during the

464

fermentation. Data are the average of three independent experiments. Error bars

465

represent ± SD.

466 467

Figure 3. Concentration of 2-furfurylthiol released by S. cerevisiae S288c (A) and

468

G20 (B). Each strain was cultured with different substrates, including furfural and

469

L-cysteine

470

average of three independent experiments. Error bars represent ± SD.

(10 mM), as well as cysteine-furfural conjugate (10 mM). Data are the

471 472

Figure 4. SDS-polyacrylamide gel of nickel affinity-purified Str3p (A) and Cys3p (B).

473

Lane M, low MW standards; Lane 1, cleared lysate; lane 2, flowthrough; Lane 3,

474

elution with 200 mM imidazole.

475


Page 24 of 34

Page 25 of 34


476

Figure 5. Effect of pH and temperature on activity (■) and stability (●) of the purified

477

Str3p (A, C) and Cys3p (B, D). Data are the average of three independent experiments.

478

Error bars represent ± SD.

479 480

Figure 6. GC/MS quantitation of enzymatic reactions with purified Str3p (A) and

481

Cys3p (B). The release of 2-furfurylthiol was quantified with headspace GC/MS in

482

reaction mixtures incubated with 3 or 6 mM cysteine-furfural-conjugated precursor.

483

Experiments were carried out with 50 µg/mL purified Str3p and Cys3p at 30 °C and at

484

pH 7.0 or 8.0 to minimize hydrolysis of the 2-furfurylthiol precursor. Data shown are

485

the means of triplicate experiments ± standard deviations of the reactions.

486

2-Furfurlthiol was not detected with empty vector control (pEASY-E1) and

487

heat-inactivated purified enzymes.

488 489

Figure 7. Mechanism of purified Str3p and Cys3p. A β-elimination reaction produces

490

2-furfurylthiol from cysteine-furfural-conjugate.



Page 26 of 34

Table 1. Strains and Plasmids Used in This Work strains and plasmids

relevant features

reference or source

plasmids pEASY-E1

Vector for protein expression, Amp R

pEASY-E1-STR3

PCR product of STR3 cloned in pEASY-E1, Amp R

this work

pEASY-E1-CYS3

PCR product of CYS3 cloned in pEASY-E1, Amp R

this work

TRANSGEN Inc., Beijing, China

strains E. coli DH5α

host for cloning

TIANGEN Inc., Beijing, China

E. coli BL21 (DE3)

host for protein expression

TIANGEN Inc., Beijing, China

E. coli BL21(STR3)

E. coli BL21 (DE3) harboring pEASY-E1-STR3, Amp R

this work

E. coli BL21(CYS3)

E. coli BL21 (DE3) harboring pEASY-E1-CYS3, Amp R

this work

S. cerevisiae S288c (ATCC 204508)

the reference strain

S. cerevisiae G20

the wild strain with the capacity of 2-furfurylthiol production; donor of STR3 and CYS3


laboratory collection laboratory collection

Page 27 of 34


Figure 1 A

B

C

D



Page 28 of 34

Figure 2 A

B


Page 29 of 34


Figure 3 A

B



Figure 4


Page 30 of 34

Page 31 of 34


Figure 5 A

B

C

D



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Figure 6 A

B


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Figure 7



For Table of Contents Only


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research 1..7 - ACS Publications - American Chemical Society

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