title: Engineering an ABC Transporter for Enhancing Resistance to Caffeine in Saccharomyces cerevisiae.
abstract: In addressing caffeine toxicity to the producing cells, engineering a transporter that can move caffeine from cytoplasm across the cell membrane to the extracellular space, thus enhancing caffeine resistance and potentially increasing the yield in yeast, is important. An ABC-transporter bfr1 from Schizosaccharomyces pombe was cloned and transformed into S. cerevisiae, resulting in enhancing caffeine resistance. Afterward, a library of randomly mutagenized bfr1 mutants through error-prone PCR was generated. One mutant was identified with drastically increased caffeine resistance (15 mg/mL). Sequencing and structural analysis illustrated that many of the mutations occurred at the cytosolic domain. Site-directed mutagenesis of these mutations confirmed at least one amino acid that conferred enhancing caffeine resistance in the mutated bfr1. These data demonstrated engineering ABC-transporters can be an efficient way to reduce product toxicity in heterologous systems.
authors.affiliation: State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University , 130 Changjiang West Road, Hefei, Anhui 230036, China.|Wuxi NewWay Biotechnology , 100 Konggang Road, Wuxi, Jiangsu 214145, China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University , 130 Changjiang West Road, Hefei, Anhui 230036, China.
authors.forename: Wei-Wei
authors.initials: WW
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University , 130 Changjiang West Road, Hefei, Anhui 230036, China.
authors.forename: Zheng-Zhu
authors.initials: ZZ
authors.lastname: Zhang
authors.affiliation: Conagen Inc., 15 DeAngelo Drive, Bedford, Massachusetts 01730, United States.|Wuxi NewWay Biotechnology , 100 Konggang Road, Wuxi, Jiangsu 214145, China.
authors.forename: Oliver
authors.initials: O
authors.lastname: Yu
keywords: ABC-transporter; caffeine; error-prone PCR; metabolic engineering