title: Modulation of heterologous expression from PBAD promoter in Escherichia coli production strains.
abstract: Promoter PBAD is frequently used for heterologous gene expression due to several advantages, such as moderately high expression levels, induction by an inexpensive and non-toxic monosaccharide L-arabinose and tight regulation of transcription, which is particularly important for expression of toxic proteins. A drawback of this promoter is all-or-none induction that occurs at subsaturating inducer concentrations. Although the overall expression level of the cell culture seems to correlate with increasing arabinose concentrations, the population is a mixture of induced and uninduced cells and with increasing arabinose concentrations, only the fraction of induced cells increases. This phenomenon is caused by autocatalytic gene expression - the expression of the arabinose transporter AraE is induced by the transported molecule. In this work the promoter PE, controlling the expression of araE, was exchanged for the stronger PBAD promoter in two Escherichia coli strains commonly used for heterologous protein production. This modification should increase a basal number of arabinose transporters in the cell wall and reduce the threshold concentration required for induction and thus reduce heterogeneity of cell population. Heterogeneity and level of expression in individual cells were analysed by flow cytometry using gfp as a reporter gene. In the strain BL21ai, the promoter exchange increased the number of induced cells at subsaturating arabinose concentrations as well as a yield of protein at saturating inducer concentration. In contrast, the modification did not improve these characteristics in RV308ai. In both strains it was possible to modulate the expression level in induced cells 3-6-fold even at subsaturating arabinose concentrations.
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia.
authors: Diana
authors: D
authors: Széliová
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia. Electronic address: krahulec@fns.uniba.sk.
authors: Ján
authors: J
authors: Krahulec
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia.
authors: Martin
authors: M
authors: Šafránek
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia.
authors: Veronika
authors: V
authors: Lišková
authors: Department of Molecular Biology, Faculty of Natural Sciences, Comenius University in Bratislava, Slovakia.
authors: Ján
authors: J
authors: Turňa
mesh_terms: D051777:AraC Transcription Factor / Q000235:genetics; D001089:Arabinose / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D051777:AraC Transcription Factor; C497371:AraC protein, E coli; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D001089:Arabinose
keywords: Arabinose; Cytometry; Escherichia coli; P(BAD) promoter; Transporter