Engineering an ABC Transporter for Enhancing Resistance to

Subscriber access provided by CORNELL UNIVERSITY LIBRARY

Article

Engineering an ABC transporter for enhancing resistance to caffeine in Saccharomyces cerevisiae Min Wang, Wei-Wei Deng, Zheng-Zhu Zhang, and Oliver Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03980 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

Journal of Agricultural and Food Chemistry

1

Engineering an ABC transporter for enhancing resistance to caffeine in

2

Saccharomyces cerevisiae

3

4

Min Wang1, 3, Wei-Wei Deng1, Zheng-Zhu Zhang1*, Oliver Yu 2, 3

5

6

1. State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural

7

University, 130 Changjiang West Road, Hefei, Anhui 230036, China

8

2. Conagen Inc., 15 DeAngelo Drive, Bedford, Massachusetts 01730, USA.

9

3. Wuxi NewWay Biotechnology, 100 Konggang Road, Wuxi, Jiangsu 214145,

10

China

11

12

*

13

Email address: [email protected] (Z.-Z. Zhang)

Corresponding author; Tel/fax: +86 551 65785471

14

1

ACS Paragon Plus Environment


15

ABSTRACT: In addressing caffeine toxicity to the producing cells, engineering a

16

transporter that can move caffeine from cytoplasm across cell membrane to

17

extracellular space, thus enhancing caffeine resistance and potentially increasing the

18

yield in yeast are important. An ABC-transporter bfr1 from Schizosaccharomyces

19

pombe was cloned and transformed into S. cerevisiae, resulting in enhancing caffeine

20

resistance. Afterwards, a library of randomly mutagenized bfr1 mutants through

21

error-prone PCR was generated. It was identified one mutant with drastically

22

increased caffeine resistance (15 mg/mL). Sequencing and structural analysis

23

illustrated that many of the mutations occurred at the cytosolic domain. Site-directed

24

mutagenesis of these mutations confirmed at least one amino acid that conferred

25

enhancing caffeine resistance in the mutated bfr1. These data demonstrated

26

engineering ABC-transporters can be an efficient way to reduce product toxicity in

27

heterologous systems.

28 29

KEYWORDS: caffeine • ABC-transporter • error-prone PCR • metabolic

30

engineering

2


Page 2 of 30

Page 3 of 30


31

INTRODUCTION

32

ATP-binding cassette transporters (ABC-transporters) form one of the largest

33

protein families in biological systems and are responsible for moving many

34

compounds across lipid bilayers.1-2 They are among the most ancient and diverse

35

proteins, and presented in almost all life forms, from bacteria to humans. Most

36

ABC-transporters are transmembrane enzymes that utilize the energy of adenosine

37

triphosphate (ATP) hydrolysis to pump various substrates across cytoplasmic

38

membranes. The substrates of these transporters include proteins, metabolic products,

39

lipids, and xenobiotic compounds.2 All ABC-transporters share a unique sequence

40

motif of their ATP-binding cassette domains that distinguish them from other

41

membrane proteins. These consensus features have been reported extensively. 1-2

42

One of the efflux carrier, P-glycoprotein, was regarded as the first member of

43

ABC-transporters in human.3 The mammalian ABC superfamily has been divided

44

into seven subfamilies (designated ABCA to ABCG) based on the relationship of

45

sequences within their NBDs (nucleotide-binding domain).4-7 Previously, six ABC

46

subfamilies in yeast were named after phylogenetic analysis. However, ABCB to

47

ABCG subfamilies were commonly accepted instead of old subfamily names MDR,

48

MRP/CFTR, ALDp, RLI, YEF3, and PDR, respectively. One thing to note here is the

49

mammalian ABCA subfamily is absolutely absent in yeast.4, 8 The PDR subfamily

3



50

plays an important role for pleiotropic drug resistance and cellular detoxification in

51

the largest subfamily in yeast.8 The best characterized PDR members in yeast

52

include PDR5/STS1/YDR1/LEM1 and SNQ2 genes.8 PDR5 was cloned as a

53

cycloheximide resistance gene and a mediating resistance gene to mycotoxins, and

54

cross-resistance to cerulenin and cycloheximide, a selective transporter of

55

glucocorticoids.8-12 SNQ2 was identified as a caffeine-resistance gene, which

56

encodes an ATP-binding cassette transporter and is highly homologous to PDR5.

57

PDR5 was also showed resistance to caffeine, while the resistance was smaller than

58

that of SNQ2.13

59

In fission yeast Schizosaccharomyces pombe, ABC-transporters play an important

60

role for resistance against xenobiotics. One of them, bfr1, is resistant to several types

61

of antibiotics, and identified as a gene partially suppressed brefeldin A

62

(BFA)-induced lethality in S. cerevisiae.14 The bfr1 belonging to PDR members,

63

encodes a novel protein of 1531 amino acids, exhibited significant homology in

64

primary and secondary structures with two reported multidrug resistance genes of S.

65

cerevisiae, Snq2 and Sts1/Pdr5/Ydr1. Though the bfr1 gene was not essential for cell

66

growth, it pumped a variety of substrates out of cells, such as cerulenin, actinomycin

67

D and cytochalasin B, in addition to BFA.15

68

Caffeine is one of the most important stimulants in everyday life. It has been used

4


Page 4 of 30

Page 5 of 30


69

extensively in beverages, nutritional supplements, and medicines.16 Many of the

70

popular energy drinks contain high levels of caffeine. However, limited numbers of

71

plant species produce this compound.17 Therefore, there is a strong interest in

72

exploring the possibility of biological production of caffeine through microbial

73

fermentation. However, caffeine is one of the most potent anti-microbial compounds

74

found in nature.18 Even though the biosynthetic pathways of caffeine can be

75

engineered in microbes, the accumulation of caffeine in these engineered strains is

76

limited by the severe toxicity of the product. The exact mechanism of caffeine

77

toxicity has not been investigated in details. It has been suspected that caffeine taken

78

up by the cells could affect DNA replication and/or transcription due to caffeine’s

79

structural similarity to nucleotides.

80

Due to the previous studies, the bfr1+ gene on a multicopy plasmid could confer

81

resistance to various antibiotics with unrelated structures, sizes, and molecular

82

targets.15 Thus, we reported a random mutagenesis study of the bfr1 transporter for

83

enhanced resistance against caffeine. The ABC-transporter bfr1 from S. pombe was

84

cloned and transformed into S. cerevisiae. We were able to confirm its function in

85

improving caffeine resistance. Furthermore, a large library of randomly mutagenized

86

bfr1 pool was generated through error-prone PCR and screened towards caffeine

87

resistance. One mutant with drastically increased caffeine resistance was confirmed

5



88

and analyzed. Structural analysis showed many of the mutations occurred at the

89

cytosolic domains. Site-directed mutagenesis of wild type bfr1 confirmed these

90

mutations enhanced caffeine resistance.

91

92

MATERIALS AND METHODS

93

Chemicals

94

Caffeine (1, 3, 7-trimethylxanthine), theophylline (1, 3-dimethylxanthine),

95

atropine and all other chemicals were purchased from Sigma-Aldrich (St Louis, MO,

96

USA), unless otherwise specified in the text.

97

Strains and plasmids

98

99

100

Escherichia coli strains, yeast strains and plasmids used in this study are shown in table 1 and 2. Media and culture conditions

101

E. coli cells were grown at 37°C in Luria Bertani (LB) medium containing 100

102

µg/mL ampicillin or 50 µg/mL kanamycin for the selection. Yeast cells were grown

103

at 30°C in standard rich medium with 2% glucose (YPD) or synthetic medium with

104

2% glucose (SD) with or without caffeine at the indicated concentration.

105

Mutant library construction and screening

106

Plasmid pDUAL-YFH1C-bfr1 containing the ORF of the bfr1 was purchased

6


Page 6 of 30

Page 7 of 30


107

from RIKEN, Japan. Gateway BP ClonaseⅡenzyme mix (Invitrogen, NY, USA)

108

catalyzes recombination between pDUAL-YFH1C-bfr1 and pDONR221, generating

109

the entry clone pENTR221-bfr1. For random mutagenesis, the Diversify PCR

110

Random Mutagenesis Kit (Clontech, Foster City, CA) was selected using condition 3

111

(the average of 2.7 mutations per 1000 bp) according to manufacturer suggested

112

protocol, with the appropriate primers to introduce mutations. The forward primer

113

5’-ATAAGAATGCGGCCGCATGAATCAAAATTCG-3’ incorporated a Not I site,

114

reverse primer 5’-CCGCTCGAGTTAACCAGTTCCGGTAATCTT-3’ incorporated

115

an Xho I site. Primers were designed by Vector NTI DNA analytical software

116

(Invitrogen, NY, USA) and synthesized by Integrated DNA Technologies (Coralville,

117

IA, USA). The PCR product of mut-bfr1 was purified by QIAquick PCR purification

118

kit (Qiagen, Germantown, MD, USA). The Not I-Xho I fragment of

119

pAG413-GPD-ccdB was replaced with the Not I-Xho I fragment containing the ORF

120

of mut-bfr1 gene. Digestions with restriction endonucleases and T4 DNA ligase were

121

used according to the manufacturer’s instruction (New England Biolabs, Ipswich,

122

MA, UK). Subsequently, E. coli Top10 cells harboring the recombinant plasmid

123

were incubated in the LB medium containing 100 µg/mL ampicillin. The mixed

124

mutant plasmids were isolated by QIAfilter-plasmid-Midi kit (Qiagen, Germantown,

125

MD, USA) and used to transform into INVSc1 by the Frozen-EZ Yeast

7



126

Transformation II kit (Orange, CA). Positive colonies were selected in liquid

127

synthetic dropout (SD) media at 30°C with all amino acids except selection marker

128

His, and contained a certain amount of caffeine. INVSc1 was transformed with the

129

mutant library of pAG413-bfr1 and directly cultured in liquid SD-His containing 10

130

mg/mL caffeine at 30°C for the first round of screening. The mixture of positive

131

colonies was plated out on SD-His agar plate containing 15 mg/mL caffeine for the

132

second round of selection.

133

134

Site-directed mutagenesis

135

All site-directed mutations were introduced according to the Quick Change

136

Site-Directed Mutagenesis Kit (Agilent Technologies, Foster City, CA) with the

137

appropriate primers introducing the mutations. For the PCR reaction, the Pfu Turbo

138

DNA polymerase (Agilent Technologies, Foster City, CA) was used to replace the

139

Phusion high-fidelity DNA polymerase (NEB) for increasing the accuracy in

140

amplification. The mutated transformants were confirmed by DNA sequencing. The

141

mutagenic oligonucleotide primers for use in this study (Table 3) were synthesized

142

by Integrated DNA Technologies.

143

Sequences and Protein structure analysis

144

The nucleotide sequences were determined by Genewiz (Germantown, MD, USA).

8


Page 8 of 30

Page 9 of 30


145

The sequence data were assembled and analyzed by Vector NTI analytical software

146

(Invitrogen, NY, USA). Protein sequences were analyzed by Cluster X software.19

147

The structure models were built based on the program I-tasser.20 ATP and caffeine

148

were docked into the built mut-bfr1 model via the program SWISDOCK.21-22

149

150

RESULTS

151

Functional expression of bfr1 increased caffeine resistance in S. cerevisiae

152

Previously, the ABC-transporter bfr1 from S. pombe has been shown to increase

153

xenobiotics resistance.23 Bfr1, whose gene was under the control of the transcription

154

factor pap1, is the major caffeine exporter in S. pombe.23 We cloned this gene into a

155

yeast expression vector pAG413-GPD-ccdB. When this vector was transformed into

156

S. cerevisiae, the cells showed higher resistance to caffeine compared to

157

non-transformed controls. As shown in Fig 1-A, caffeine was cytotoxic to the yeast

158

cells, and inhibited their growth at concentration of 4 mg/mL and 8 mg/mL. The time

159

course assay showed in Fig1-B, indicated that INVSc1 cells with the presence of

160

bfr1 conferred high levels of caffeine resistance (8 mg/mL). The cells showed

161

significantly higher density when 8 mg/mL of caffeine was added to the media

162

(OD600=0.93).

163

Mutagenesis of bfr1 and the library transformation

9



164

The mutant library was constructed using error-prone PCR with the Diversify

165

PCR Random Mutagenesis Kit (Clontech, Foster City, CA) at buffer condition 3.

166

The wild type bfr1 gene was used as the template. The reaction, including nucleotide

167

analogues, mutant polymerases, and high concentration of Mn2+, was scaled up to

168

produce enough pooled DNA for large scale yeast transformation. The

169

pAG413-GPD-ccdB plasmid, supports bacterial and yeast replication, was selected

170

as the expression vector. The Not I and Xho I digested mutated bfr1 fragments and

171

vector were ligated over night. In order to obtain a collection of 106 mutants for the

172

mutant library, a total of 60 mg of the recombinant plasmids were extracted using

173

QIA filter Plasmid Midi Kit (Qiagen, Germantown, MD, USA).

174

For the screening of positive colonies, a liquid SD-His containing 10 mg/mL

175

caffeine was used in culturing INVSc1 which was transformed with the mutant

176

library of pAG413-bfr1 at the first round; the mixture of positive colonies was plated

177

out on SD-His agar plate containing 15 mg/mL caffeine for the second round of

178

selection. After two rounds of selection, at least 4 independent mutants were

179

generated.

180

Mutagenized bfr1 has increased caffeine resistance

181

In screening of the mutagenized library on increased caffeine-containing medium,

182

one transformant, named mut-bfr1-B, was found to have significantly higher caffeine

10


Page 10 of 30

Page 11 of 30


183

resistance. As shown in Figure 2, the mutant bfr1-B provided the cells drastically

184

higher caffeine resistance in both liquid culture and sequential dilution spots on solid

185

plates containing 15 mg/mL of caffeine. It was also observed that the mutant strain

186

was resistant to caffeine up to 25 mg/mL (data not shown). Thus, the mut-bfr1-B was

187

chosen for further analysis.

188

Modeling of the mutations on bfr1

189

The plasmid pAG413-bfr1-B was isolated from yeast, amplified in E. coli Top10,

190

and sequenced. We observed that there were many mutations in bfr1-B sequence (the

191

overall mutations information as shown in Table 4). The transcribed amino acid

192

sequence was aligned with original bfr1 and it was confirmed that there are 11 amino

193

acid mutations involved (Table 4).

194

When a structural model was constructed to highlight the locations of the

195

mutations, based on existing ABC-transporter models, we found some of the

196

mutations (are located at nucleotide-binding-domain (NBD), instead of the putative

197

caffeine binding domain (Figure 3). The directed mutagenesis was carried out to

198

confirm these mutations and their function in caffeine resistance.

199

Site-directed mutagenesis of the bfr1

200

Three amino acids were selected according to the predicted structural model. And

201

the site-directed mutagenesis was carried out using the wild type bfr1 gene as the

11



202

template. The three-dimensional model of bfr1 indicated that amino acid residues at

203

positions S36 (named as site-mut1) and D340 (named as site-mut2) located in the

204

NBDs, while Y497 (named as site-mut3) located in the TMDs (Figure 3). The

205

transgenic S. cerevisiae carrying the bfr1 mutants were tested on SD-His agar plate

206

containing 10 mg/mL caffeine. Two of the mutations, site-mut1 and site-mut3,

207

showed similar caffeine sensitivity as the wild type bfr1 strain, suggesting these

208

mutations individually did not increase the caffeine resistance.

209

However, the third mutation site-mut2 enhanced caffeine resistance. As shown in

210

Figure 4, although the resistance was not as strong as the original bfr1-B mutant,

211

who carries multiple mutations, site-mut2 had higher resistance to caffeine than wild

212

type bfr1. As illustrated in the plating screening, the sequential spots of site-mut2

213

mutant were less resistant to caffeine than the mut-bfr1-B at the concentration of 10

214

mg/mL, while the wild type bfr1 bearing strain did not grow at all. The single colony

215

of site-mut2 mutant was also inoculated into liquid SD-His medium and confirmed

216

with 15 mg/mL of caffeine (data not shown).

217

The mutant bfr1-B showed more sensitivity to other secondary metabolites

218

Since bfr1-B showed increased resistance to caffeine, we were interested whether

219

the same mutant increased the resistance to other similar metabolites, such as

220

compounds with similar structural and pharmacological functions. Thus, the growth

12


Page 12 of 30

Page 13 of 30


221

profile of S. cerevisae INVSc1, S. cerevisae INVSc1 (pAG413-bfr1), and S.

222

cerevisae INVSc1 (pAG413-bfr1-B) with theophylline (similar structural compound,

223

similar pharmacological function) and atropine (different structural compound) were

224

compared, as shown in Figure 5. Surprisingly, the bfr1 mutant did not increase

225

resistance to these compounds, which actually caused more sensitivity than the wild

226

type strain. The bfr1-B mutant is more sensitive to both theophylline and atropine.

227

228

DISCUSSION

229

A common challenge in synthetic biology is the toxicity issues related to excessive

230

metabolite accumulations arising from metabolic engineering. Over-production of

231

targeted compounds is one of the most important goals of bio-manufacturing in

232

general. However, except for a few compounds, accumulation of the products, in

233

some occasions, even the feeding of large amount of metabolic substrate can cause

234

toxicity to the producing cells. The ATP-binding cassette transporters, involved in

235

the transportation of a variety of molecules across the cellular membranes, play a

236

key role in detoxification of foreign compounds.

237

In S. pombe, acting downstream of stress-activated kinase Pap1, some transporters

238

are significantly induced, including bfr1/hba2.24 Through genome-wide screen, cell

239

lacking both hba2/bfr1 and pap1 was very sensitive to caffeine in S. pombe.23 The

13



240

bfr1, belonging to PDR members, exhibited significant homology in primary and

241

secondary structures with two reported multidrug resistance genes of S. cerevisiae,

242

Snq2 and Sts1/Pdr5/Ydr1. Snq2p is functional homologous to Pdr5p, which is also a

243

plasma membrane ABC transporter. After the investigation of the function roles of

244

Snq2p and Pdr5p, it was demonstrated that Snq2p and Pdr5p mediate caffeine efflux

245

and resistance in S. cerevisiae.

246

coupled with a low-copy number plasmid in a global transcription machinery

247

engineering studies to investigate the role of bfr1 in detoxification.25 Based on their

248

initial discoveries, pAG413-GPD-ccdB, a low-copy number CEN-based plasmid was

249

selected as an expression vector in S. cerevisiae for enhancing caffeine resistance

250

and tried to increase the yield of engineered caffeine biosynthetic pathway

251

afterwards. It was confirmed that expression of bfr1 conferred to increase caffeine

252

resistance as expected. Furthermore, error-prone PCR was employed to further

253

engineer the bfr1 gene for higher caffeine resistance. The resulting mutant

254

pAG413-bfr1-B provided higher caffeine resistance of more than 15mg/mL, and

255

even 25 mg/mL. Besides, in the presence of 20 mM caffeine in the previous report,

256

YPH250 cells harboring Yep24-PDR5 (PDR5-over-expressed cells) grew very little,

257

while YPH250 cells harboring Yep24-SNQ2 (SNQ2-overexpressing cells) grew

258

significantly. 13

13

The group used a strong constitutive promoter

14


Page 14 of 30

Page 15 of 30


259

The observed mutations led to the higher caffeine resistance were confirmed.

260

Site-directed mutagenesis was performed targeting three of the mutants and analyzed

261

their corresponding caffeine resistance. Among the three mutation sites tested,

262

site-mut2 increased caffeine resistance, while the other two mutations did not yield

263

elevated caffeine resistance. Since site-mut2 conferred aspartic acid residue

264

converted to glycine, which might cause the structural and functional change of

265

NBDs, the cells harboring site-mut2 gene product seemed to strongly enhanced the

266

ATP binding and hydrolysis. Compare to pAG413-bfr1-B, the site-mut2 mutant was

267

less resistance to caffeine; the results suggested that some of the mutations are

268

involved in direct caffeine resistance, which may involve some synergistic effects

269

among different mutations.

270

In exploring whether the bfr1 confer to resistance of wider group of compound

271

similar to caffeine, we investigated bfr1/bfr1-B could increase resistance to other

272

chemicals like theophylline and atropine or not. Since theophylline and caffeine are

273

similar in chemical nature, molecular weight, and pharmacological function; while

274

atropine is more distinct from caffeine. Interestingly we found the cells bearing both

275

the bfr1 and bfr1-B exhibited higher sensitivity towards these two alkaloids than the

276

wild type strain. Thus, we proposed that the transporter bfr1/bfr1-B seem to have

277

stringent substrate specificity and laid a burden on cells for other detoxification

15



278

functions.

279

280

FUNDING SOURCES

281

This study was supported by the National Natural Science Foundation of China

282

(NSFC) project 31570692, the Changjiang Scholars and Innovative Research Team

283

in University (IRT_15R01), Anhui Major Demonstration Project for Leading Talent

284

Team on Tea Chemistry and Health, National Modern Agriculture Technology

285

System (CARS-23), and Chinese National 863 Project (Award 2013AA102801 to

286

O.Y.)

287

288

References

289

(1) Higgins, C.F. ABC transporters: from microorganisms to man. Annu Rev Cell

290

Physiol. 1992, 8, 67—113.

291

(2) Rea, P.A., Li, Z.S., Lu, A.Y.P, Drozdowicz, Y.M., Martinoia, E. From vacuolar

292

GS-X pumps to multispecific ABC transporters. Annu Rev Plant Physiol Plant Mol

293

Biol. 1998, 49, 727—760.

294

(3) Juliano, R.L., Ling V. A surface glycoprotein modulating drug permeability in

295

Chinese hamster ovary cell mutants. Biochim Biophys Acta. 1976, 455, 152—162.

296

(4) Paumi, C.M., Chuk, M., Snider, J., Stagljar, I., Michaelis, S. ABC transporters in

16


Page 16 of 30

Page 17 of 30


297

Saccharomyces cerevisiae and their interactors: new technology advances the

298

biology of the ABCC (MRP) subfamily. Microbiol Mol Biol Rev. 2009, 73, 577—93.

299

(5) Dean, M. The genetics of ATP-binding cassette transporters. Methods Enzymol.

300

2005, 400, 409—429.

301

(6) Dean, M., Rzhetsky, A., Allikmets, R. The human ATP-binding cassette (ABC)

302

transporter superfamily. Genome Res. 2001, 11, 1156—1166.

303

(7) Dean, M., Allikmets, R. Complete characterization of the human ABC gene

304

family. J Bioenerg Biomembr. 2001, 33, 475—479.

305

(8) Bauer, B.E., Wolfger, H., Kuchler, K. Inventory and function of yeast ABC

306

proteins: about sex, stress, pleiotropic drug and heavy metal resistance. Biochim

307

Biophys Acta. 1999, 1461, 217—236.

308

(9) Balzi, E., Wang, M., Leterme, S, Van Dyck, L, Goffeau, A. PDR5, a novel yeast

309

multidrug-resistance-conferring transporter controlled by the transcription regulator PDR1. J

310

Biol Chem. 1994, 269, 2206—2214.

311

(10) Bissinger, P.H., Kuchler, K. Molecular cloning and expression of the S.

312

cerevisiae STS1 gene product. J Biol Chem. 1994, 269, 4180—4186.

313

(11) Hirata, D., Yano, K., Miyahara, K., Miyakawa, T. Saccharomyces cerevisiae

314

YDR1, which encodes a member of the ATP-binding cassette (ABC) superfamily, is

315

required for multidrug resistance. Curr Genet. 1994, 26, 285—294. 17



316

(12) Kralli, A., Bohen, S.P., Yamamoto, K.R. LEM1, an ATP-binding-cassette

317

transporter, selectively modulates the biological potency of steroid hormones. Proc

318

Natl Acad Sci USA. 1995, 92, 4701—4705

319

(13) Tsujimoto, Y., Shimizu, Y., Otake, K., Nakamura, T., Okada, R., Miyazaki, T.,

320

Watanabe, K. Multidrug resistance transporter Snq2p and Pdr5p mediate caffeine

321

efflux in Saccharomyces cerevisiae. Biosci Biotechnol Biochem. 2015, 79,

322

1103—1110

323

(14) Jackson, C.L., Kepes, F. BFRl, a multicopy suppressor of brefeldin A-induced

324

lethality, is implicated in secretion and Nuclear segregation in Saccharomyces

325

cerevisiae. Genetics. 1994, 137, 423—437.

326

(15) Nagao, K., Taguchi, Y., Arioka, M., Kadokura, H., Takatsuki, A., Yoda, K.,

327

Yamasaki, M. bfr1+, a novel gene of Schizosaccharomyces pombe which confers

328

brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily.

329

J Bacteriol. 1995, 177, 1536—1543.

330

(16) Matissek, R. Evaluation of xanthine derivatives in chocolate - nutritional and

331

chemical aspects. Z Lebensm Unters Forsch. 1997, 205, 175—84.

332

(17) Suzuki, T., Ashihara, H., Waller, G.R. Purine and purine alkaloid metabolism in

333

Camellia and Coffea plants. Phytochem. 1992, 31, 2575—2584.

334

(18) Esimone, C.O., Okoye, F.B., Nworu, C.S., Agubata, C.O. In vitro interaction

18


Page 18 of 30

Page 19 of 30


335

between caffeine and some penicillin antibiotics against staphylococcus aureus. Trop

336

J Pharm Res. 2008, 7, 969—974.

337

(19) Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G. The

338

clustal_x windows interface: flexible strategies for multiple sequence alignment

339

aided by quality analysis tools. Nucleic Acids Res. 1997, 25, 4876—4882.

340

(20) Zhang Y. I-TASSER server for protein 3D structure prediction. BMC

341

Bioinformatics. 2008, 9, 40.

342

(21) Grosdidier A., Zoete V., Michielin O. SwissDock, a protein-small molecule

343

docking web service based on EADock DSS. Nucleic Acids Res. 2011, 39,

344

W270—277.

345

(22) Jin, L., Bhuiya, M. W., Li, M., Liu, X., Han, J., Deng, W., Wang, M., Yu, O.,

346

Zhang, Z. Metabolic engineering of Saccharomyces cerevisiae for caffeine and

347

theobromine production. Plos One. 2014, 9, e105368—105368.

348

(23) Calvo, I.A., Gabrielli, N,, Iglesias-Baena, I., García-Santamarina, S., Hoe, K.L.,

349

Kim, D.U., Sansó, M., Zuin, A., Pérez, P., Ayté, J., Hidalgo, E. Genome-wide screen

350

of genes required for caffeine tolerance in fission yeast. PLoS One. 2009, 4, e6619.

351

(24) Chen, D., Wilkinson, C.R.M., Watt, S., Penkett, C.J., Toone, W.M., Jones, N.,

352

Bähler, J. Multiple pathways differentially regulate global oxidative stress responses

353

in fission yeast. Mol Biol Cell. 2008, 19, 308—317.

19



354

(25) Alper, H., Stephanopoulos, G. Global transcription machinery engineering: a

355

new approach for improving cellular phenotype. Metab Eng. 2007, 9, 258—267.

356

20


Page 20 of 30

Page 21 of 30


357

Legends for Figures

358

Figure 1. The time course growth of bfr1 strain.

359

A, Growth curves of wild-type S. cerevisiae INVSc1 in different concentrations of

360

caffeine in liquid SD media; B, Growth curves of WT-INVSc1 and WT-

361

pAG413-bfr1 in liquid SD (-His) media containing 8 mg/mL of caffeine.

362

Figure 2. Growth phenotypes of mutant bfr1 strain.

363

A, Cultures of WT-INVSc1, WT-pAG413-bfr1 and mut-pAG413-bfr1-B were

364

treated with 15 mg/mL of caffeine in liquid SD-His media. Growth was monitored

365

by measuring OD600. B, Strains of WT-INVSc1, WT-pAG413-bfr1 and

366

mut-pAG413-bfr1-B were grown in liquid SD-His media and diluted to an OD600 of

367

0.6 in media in duplicate. Serial dilutions (1:5:52:53:54) were spotted on SD-His plate

368

containing 15 mg/mL of caffeine.

369

Figure 3. Structure modeling of the mut-bfr1.

370

Nucleotide binding domains (NBDs) and transmembrane domains (TMDs) are

371

labeled. ATP and caffeine are represented by sphere. Mutant amino acid residues are

372

labeled in yellow sphere.

373

Figure 4. Mutation site-mut2 showed enhanced caffeine resistance.

374

Strains of WTpAG413-bfr1, mut-pAG413-bfr1-B and site-mut2 were grown in

21



375

liquid SD-His media and diluted to an OD600 of 0.6 in media in triplicates. Serial

376

dilutions were spotted on SD-His plate containing 10 mg/mL of caffeine.

377

Figure 5. Comparison of S. cerevisiae resistance to theophylline and atropine

378

with bfr1 and its mutant.

379

Cultures of INVSc1, INVSc1 (pAG413-bfr1) and INVSc1 (pAG413-bfr1-B) were

380

treated with the indicated concentration of theophylline or atropine. Growth was

381

monitored by measuring OD600. A, 6 mg/mL of theophylline; B, 4.4 mg/mL of

382

atropine.

22


Page 22 of 30

Page 23 of 30


Table 1. Strains used in this study. Srtain

Genotype

Source

E. coli TOP10

F-mcrA ∆(mrr-hsdRMS-mcrBC)φ80lacZ∆M15

Invitrogen

S. cerevisiae

∆lacX74 recA1 araD139 ∆(araleu)7697 galU galK rpsL (StrR) endA1 nupG MATa ;his3D1 leu2 trp1-289 ura3-52 Invitrogen

INVSc1

Table 2. Plasmids used in this study. Plasmid

Vector; Insert

Source

pDUAL-YFH1c-bfr1 pDUAL-YFH1c; bfr1 pENTR-bfr1 pDONR221; bfr1 pAG413-GPD-bfr1 pAG413-GPD-ccdB; bfr1

RIKEN, Japan This study This study

Table 3. Mutagenic oligonucleotide primers used in this study. The desired mutations in the primers are underlined. Primers

Description

F-site-mut1 R-site-mut1 F-site-mut2 R-site-mut2

5’-TCTAATTCCTCTGATCATTTCGAGGATCCTTCTTCG-3’ 5’-TCTAAAGACTCGTCAACATTCGAAGAAGGATCCTCG-3’ 5’-AATAGTACTCGTGGTTTGGGCTCTAGTAC-3’ 5’-AACTCGAAAGCCGTACTAGAGCCCAAACCAC-3’

F-site-mut3 R-site-mut3

5’-ACTACCAAGCATGAGCTCCATCGTCAAAGTG-3’ 5’-ACTTTGACGATGGAGCTCATGCTTGGTAGTG-3’

23



Page 24 of 30

Table 4. Mutational sites from bfr1 sequencing in colony B. Mutant nucleotides positions

Nucleotide changes bfr1

Amino acids

106 1019 1489 2487

Tct gAc Tat

Cct gGc Cat

gcT

gcC

2933

tTg Tca

tAg Cca

3001 3028 3275 3462 3594 3711 3857 3908

positions

bfr1-B

Agc gAt tgG

Tgc gGt tgA

ccT ttT aAg gTa

ccC ttG aGg gCa

Amino acid changes bfr1

bfr1-B

36 340

Ser Asp

Pro Gly

497 829

Tyr Ala

His Ala

978

Leu

Stop

1001 1010 1092 1154 1198 1237 1286 1303

Ser Ser Asp Trp Pro Phe Lys Val

Pro Cys Gly Stop Pro Leu Arg Ala

24


Page 25 of 30


Figure 1

25



Figure 2

26


Page 26 of 30

Page 27 of 30


Figure 3

27



Figure 4

28


Page 28 of 30

Page 29 of 30


Figure 5

29



Graphic for table of contents

30


Page 30 of 30

Engineering an ABC Transporter for Enhancing Resistance to

Recommend Documents