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

摘要

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 degrees C. To isolate a mutant TliDEF capable of secreting TliA at 35 degrees 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 WE, tliF and tliA in Escherichia con. Among approximately 10,000 colonies of the tliD library, we selected one colony that formed transparent halo on LB-tributyrin plates at 35 degrees C. At the growth temperature of 35 degrees 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 degrees C, 25 degrees C and 30 degrees 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. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.