Hydroxy-mandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122–126 were involved in its PobR ligand specificity. These results showed the effectiveness of directed evolution in switching the ligand specificity of a biosensor and improving HMA production.
Hongxuan Du,Yaoyao Liang,Jianing Li,Xinyao Yuan,Fenglin Tao,Chengjie Dong,Zekai Shen,Guangchao Sui,Pengchao Wang
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.
危险品化学品经营许可证（带存储）