Directed Evolution of 4-Hydroxyphenylpyruvate Biosensors Based on a Dual Selection System

Biosensors based on allosteric transcription factors have been widely used in synthetic biology. In this study, we utilized the Acinetobacter ADP1 transcription factor PobR to develop a biosensor activating the PpobA promoter when bound to its natural ligand, 4-hydroxybenzoic acid (4HB). To screen for PobR mutants responsive to 4-hydroxyphenylpyruvate(HPP), we developed a dual selection system in E. coli. The positive selection of this system was used to enrich PobR mutants that identified the required ligands. The following negative selection eliminated or weakened PobR mutants that still responded to 4HB. Directed evolution of the PobR library resulted in a variant where PobR^W177R was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobR^WT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.