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

ABSTRACT: 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.

SUBMITTER: Du H

PROVIDER: S-EPMC10855707 | biostudies-literature | 2024 Jan

REPOSITORIES: biostudies-literature

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

Du Hongxuan H Liang Yaoyao Y Li Jianing J Yuan Xinyao X Tao Fenglin F Dong Chengjie C Shen Zekai Z Sui Guangchao G Wang Pengchao P

International journal of molecular sciences 20240126 3

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 ...[more]

PMID: 38338812

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Project description:Directed evolution is a powerful tool that can bypass gaps in our understanding of the sequence-function relationship of proteins and still isolate variants with desired activities, properties, and substrate specificities. The rise of directed evolution platforms for polymerase engineering has accelerated the isolation of xenobiotic nucleic acid (XNA) synthetases and reverse transcriptases capable of processing a wide array of unnatural XNAs which have numerous therapeutic and biotechnological applications. Still, the current generation of XNA polymerases functions with significantly lower efficiency than the natural counterparts and retains a significant level of DNA polymerase activity which limits their in vivo applications. Although directed evolution approaches are continuously being developed and implemented to improve XNA polymerase engineering, the field lacks an in-depth analysis of the effect of selection parameters, library construction biases and sampling biases. Focusing on the directed evolution pipeline for DNA and XNA polymerase engineering, this work sets out a method for understanding the impact of selection conditions on selection success and efficiency. We also explore the influence of selection conditions on fidelity at the population and individual mutant level. Additionally, we explore the sequencing coverage requirements in directed evolution experiments, which differ from genome assembly and other -omics approaches. This analysis allowed us to identify the sequencing coverage threshold for the accurate and precise identification of significantly enriched mutants. Overall, this study introduces a robust methodology for optimizing selection protocols, which effectively streamlines selection processes by employing small libraries and cost-effective NGS sequencing. It provides valuable insights into critical considerations, thereby enhancing the overall effectiveness and efficiency of directed evolution strategies applicable to enzymes other than the ones considered here.

Dataset Information

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

Publications

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

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