Abstract
Directed evolution has been proven as a powerful tool for developing proteins and strains with novel or enhanced features. In this study, a dual selection system was designed to tune the binding specificity of a transcription factor to a particular ligand with the ampicillin resistance gene amp (ON selection) as the positive selection marker and the levansucrase gene sacB (OFF selection) as the negative selection marker. It was applied to the lead responsive transcription factor PbrR in a whole-cell lead biosensor previously constructed in our lab (Jia et al. in Fems Microbiol Lett 365:fny157, 2018). After multiple rounds of ON–OFF selection, two mutants with higher specificity for lead were selected. Structural analysis revealed that the mutation C134 located on the metal-binding loop at the C-terminal of PbrR is likely associated with the enhanced binding to both lead and cadmium. The double mutations D64A and L68S close to the metal-binding residue C79 may lead to the reduced binding specificity toward zinc ions. This dual selection system can be applied to engineer the specificity of other transcription factors and provide fine-tuned tools to synthetic biology.
Highlights
In recent years, directed evolution has been widely used for the development and the improvement of enzymes and proteins (Currin et al 2015)
During the ON selection, cells with PbrR mutants that have strong response to the lead ions and activate the expression of amp could survive in presence of ampicillin, while weak binding mutants led to cell death (Fig. 1)
Metal-binding transcription factors, such as those in the MerR family in response to divalent ions, are commonly used in whole-cell heavy metal biosensors, and their specificity determines the accuracy of the sensors
Summary
In recent years, directed evolution has been widely used for the development and the improvement of enzymes and proteins (Currin et al 2015). The key to directed evolution lies in the construction of the mutant library and the efficiency of the screening process. Developing efficient screening strategies has become the bottleneck of protein directed evolution technology (Cadwell and Joyce 1992; Stemmer 1994; Zhao et al 1998). Jia et al AMB Expr (2020) 10:67 will be selected using the negative marker, the levansucrase gene sacB, and only the ones that do not respond to these inducers can survive. By alternating these ON– OFF selection steps for multiple cycles, mutant regulators only in response to the target inducer are expected to be obtained. This strategy can be applied to enhance the specificity of transcription factor based biosensors in a way that it strengthens the signal of the biosensors in response to the target analyte and minimizes the noise
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