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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
- Eom, Gyeong Tae, Oh, Joon Young, Park, Ji Hyun, Lim, Hye Jin, Lee, So Jeong, Kim, Eun Young, Choi, Ji-Eun, Jegal, Jonggeon, Song, Bong Keun, Yu, Ju-Hyun, Song, Jae Kwang
- Journal of bioscience and bioengineering 2016 v.122 no.3 pp. 283-286
- ABC transporters, Escherichia coli, Pseudomonas fluorescens, amino acids, carboxylic ester hydrolases, mutagenesis, mutants, point mutation, polymerase chain reaction, secretion, temperature
- 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.