title: Alleviation of temperature-sensitive secretion defect of Pseudomonas fluorescens ATP-binding cassette (ABC) transporter, TliDEF, by a change of single amino acid in the ABC protein, TliD.
abstract: An ABC transporter, TliDEF, from Pseudomonas fluorescens SIK W1, mediates the secretion of its cognate lipase, TliA, in a temperature-dependent secretion manner; the TliDEF-mediated secretion of TliA was impossible at the temperatures over 33°C. To isolate a mutant TliDEF capable of secreting TliA at 35°C, the mutagenesis of ABC protein (TliD) was performed. The mutated tliD library where a random point mutation was introduced by error-prone PCR was coexpressed with the wild-type tliE, tliF and tliA in Escherichia coli. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35°C. At the growth temperature of 35°C, the selected mutant TliD showed 1.75 U/ml of the extracellular lipase activity, while the wild-type TliDEF did not show any detectable lipase activity in the culture supernatant of E. coli. Moreover, the mutant TliD also showed higher level of TliA secretion than the wild-type TliDEF at other culture temperatures, 20°C, 25°C and 30°C. The mutant TliD had a single amino acid change (Ser287Pro) in the predicted transmembrane region in the membrane domain of TliD, implying that the corresponding region of TliD was important for causing the temperature-dependent secretion of TliDEF. These results suggested that the property of ABC transporter could be changed by the change of amino acid in the ABC protein.
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea; Center for Industrial Chemical Biotechnology, Korea Research Institute of Chemical Technology (KRICT), 45 Jongga-ro, Jung-gu, Ulsan 681-802, Republic of Korea.
authors.forename: Gyeong Tae
authors.initials: GT
authors.lastname: Eom
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Joon Young
authors.initials: JY
authors.lastname: Oh
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Park
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea; Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 305-764, Republic of Korea.
authors.forename: Hye Jin
authors.initials: HJ
authors.lastname: Lim
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: So Jeong
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Ji-Eun
authors.initials: JE
authors.lastname: Choi
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea; Center for Industrial Chemical Biotechnology, Korea Research Institute of Chemical Technology (KRICT), 45 Jongga-ro, Jung-gu, Ulsan 681-802, Republic of Korea.
authors.forename: Jonggeon
authors.initials: J
authors.lastname: Jegal
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea; Center for Industrial Chemical Biotechnology, Korea Research Institute of Chemical Technology (KRICT), 45 Jongga-ro, Jung-gu, Ulsan 681-802, Republic of Korea.
authors.forename: Bong Keun
authors.initials: BK
authors.lastname: Song
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Ju-Hyun
authors.initials: JH
authors.lastname: Yu
authors.affiliation: Research Center for Bio-based Chemistry, Korea Research Institute of Chemical Technology (KRICT), 141 Gajeong-ro, Yuseong-gu, Daejeon 305-600, Republic of Korea; Department of Green Chemistry and Environmental Biotechnology, Korea University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea. Electronic address: ajee@krict.re.kr.
authors.forename: Jae Kwang
authors.initials: JK
authors.lastname: Song
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000378:metabolism; D017354:Point Mutation; D011551:Pseudomonas fluorescens / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D018528:ATP-Binding Cassette Transporters; D000596:Amino Acids; D001426:Bacterial Proteins; D003470:Culture Media; D008049:Lipase
keywords: ABC protein; ATP-binding cassette (ABC) transporter; Lipase; Pseudomonas fluorescens; Random mutagenesis; Temperature-dependent secretion