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

Author:

Du Hongxuan123,Liang Yaoyao123,Li Jianing12,Yuan Xinyao12,Tao Fenglin12,Dong Chengjie24,Shen Zekai5,Sui Guangchao1234ORCID,Wang Pengchao1234

Affiliation:

1. School of Life Science, Northeast Forestry University, Harbin 150040, China

2. NEFU-China iGEM Team, Northeast Forestry University, Harbin 150040, China

3. Key Laboratory for Enzyme and Enzyme-Like Material Engineering of Heilongjiang, College of Life Science, Northeast Forestry University, Harbin 150040, China

4. Aulin College, Northeast Forestry University, Harbin 150040, China

5. School of Pharmacology, China Pharmaceutical University, Nanjing 210009, China

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 PobRW177R was 5.1 times more reactive to 4-hydroxyphenylpyruvate than PobRWT. Overall, we developed an efficient dual selection system for directed evolution of biosensors.

Funder

Fundamental Research Funds for the Central Universities

National Natural Science Foundation of China

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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