Engineering an ABC Transporter for Enhancing Resistance to

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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

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Engineering an ABC transporter for enhancing resistance to caffeine in

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Saccharomyces cerevisiae

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

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

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

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

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

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China

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*

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

Corresponding author; Tel/fax: +86 551 65785471

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

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transporter that can move caffeine from cytoplasm across cell membrane to

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extracellular space, thus enhancing caffeine resistance and potentially increasing the

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

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

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

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

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

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

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

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enhancing caffeine resistance in the mutated bfr1. These data demonstrated

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

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heterologous systems.

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

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engineering

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INTRODUCTION

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

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protein families in biological systems and are responsible for moving many

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

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

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

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triphosphate (ATP) hydrolysis to pump various substrates across cytoplasmic

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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that of SNQ2.13

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

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

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

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

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

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

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

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

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D and cytochalasin B, in addition to BFA.15

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

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

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

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

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exploring the possibility of biological production of caffeine through microbial

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

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

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

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

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

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

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structural similarity to nucleotides.

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

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

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

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

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

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

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

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resistance. One mutant with drastically increased caffeine resistance was confirmed

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

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

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mutations enhanced caffeine resistance.

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MATERIALS AND METHODS

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Chemicals

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

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

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USA), unless otherwise specified in the text.

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Strains and plasmids

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

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

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

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

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

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Mutant library construction and screening

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

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

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

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

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

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

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

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

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reverse primer 5’-CCGCTCGAGTTAACCAGTTCCGGTAATCTT-3’ incorporated

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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second round of selection.

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Site-directed mutagenesis

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

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

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

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

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

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

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

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by Integrated DNA Technologies.

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Sequences and Protein structure analysis

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

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

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

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

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

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RESULTS

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Functional expression of bfr1 increased caffeine resistance in S. cerevisiae

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

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

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

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

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

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

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

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

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

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

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(OD600=0.93).

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Mutagenesis of bfr1 and the library transformation

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

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

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

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

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produce enough pooled DNA for large scale yeast transformation. The

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

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

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

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

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

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

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

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

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

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

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generated.

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Mutagenized bfr1 has increased caffeine resistance

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

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

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

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

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

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

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chosen for further analysis.

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Modeling of the mutations on bfr1

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

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

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

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

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acid mutations involved (Table 4).

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

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

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

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

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confirm these mutations and their function in caffeine resistance.

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Site-directed mutagenesis of the bfr1

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

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

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

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

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

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

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

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

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mutations individually did not increase the caffeine resistance.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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DISCUSSION

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

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metabolite accumulations arising from metabolic engineering. Over-production of

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

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

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

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

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

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key role in detoxification of foreign compounds.

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

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

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

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

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

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

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

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

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and resistance in S. cerevisiae.

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

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

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

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

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and tried to increase the yield of engineered caffeine biosynthetic pathway

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

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

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

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

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

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

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

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significantly. 13

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The group used a strong constitutive promoter

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

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

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

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

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

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

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

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

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

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

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among different mutations.

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

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

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

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

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

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

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

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

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functions.

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FUNDING SOURCES

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

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

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

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Team on Tea Chemistry and Health, National Modern Agriculture Technology

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

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O.Y.)

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

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

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

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

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

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

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

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

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

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

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Legends for Figures

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Figure 1. The time course growth of bfr1 strain.

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

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

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

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Figure 2. Growth phenotypes of mutant bfr1 strain.

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

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

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

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

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

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containing 15 mg/mL of caffeine.

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Figure 3. Structure modeling of the mut-bfr1.

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

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

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labeled in yellow sphere.

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

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

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

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

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

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with bfr1 and its mutant.

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

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

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

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atropine.

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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’

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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

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