Directed evolution of the PobR allosteric transcription factor to generate a biosensor for 4-hydroxymandelic acid

• Main Authors: Chen, F. (Author), Dong, Y. (Author), Fu, H. (Author), Guo, S. (Author), Liang, Y.Y (Author), Luo, J. (Author), Su, K. (Author), Sui, G. (Author), Wang, P. (Author), Wang, Z. (Author), Yang, C. (Author), Zhang, B. (Author), Zhang, Y. (Author)
• Format: Article
• Language: English
• Published: Springer Science and Business Media B.V. 2022
• Subjects: 4-hydroxybenzoic acids; Acid production; Biosensors; Directed evolution; Error prone PCR; Hydroxymandelic acid; Hydroxy-mandelic acid; Ligand specificity; Ligands; Mandelic acid; Mutant libraries; Organic acids; Pobr; PobR; Transcription; Transcription factors
• Online Access: View Fulltext in Publisher

 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. © 2022, The Author(s), under exclusive licence to Springer Nature B.V.