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 theAcinetobacterADP1 transcription factor PobR to develop a biosensor activating the PpobApromoter 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 inE. 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 PobRW177Rwas 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobRWT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.