Computer-Aided Rational Engineering of Signal Sensitivity of Quorum Sensing Protein LuxR in a Whole-Cell Biosensor
| Autor: | Shuxia Liu, Tao Wang, Jinyu Li, Tuoyu Liu, Ruicun Liu, Shan Yang, Yajun Song, Yue Teng, Yulu Chen, Cheng Chen, Yingtan Zhuang, Ruifu Yang, Yujun Cui |
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| Rok vydání: | 2021 |
| Předmět: |
gene circuit
QH301-705.5 biosensor medicine.disease_cause Biochemistry Genetics and Molecular Biology (miscellaneous) Biochemistry Molecular mechanics Green fluorescent protein medicine Molecular Biosciences Biology (General) Surface plasmon resonance Molecular Biology Escherichia coli Transcription factor Original Research signal sensitivity Chemistry Rational design quorum sensing food and beverages biochemical phenomena metabolism and nutrition Quorum sensing bacteria LuxR Biosensor rational protein engineering |
| Zdroj: | Frontiers in Molecular Biosciences, Vol 8 (2021) Frontiers in Molecular Biosciences |
| ISSN: | 2296-889X |
| DOI: | 10.3389/fmolb.2021.729350 |
| Popis: | LuxR, a bacterial quorum sensing-related transcription factor that responds to the signaling molecule 3-oxo-hexanoyl-homoserine lactone (3OC6-HSL). In this study, we employed molecular dynamics simulation and the Molecular Mechanics Generalized Born Surface Area (MM-GB/SA) method to rationally identify residues in Vibrio fischeri LuxR that are important for its interaction with 3OC6-HSL. Isoleucine-46 was selected for engineering as the key residue for interaction with 3OC6-HSL-LuxR-I46F would have the strongest binding energy to 3OC6-HSL and LuxR-I46R the weakest binding energy. Stable wild-type (WT) LuxR, I46F and I46R variants were produced in Escherichia coli (E. coli) in the absence of 3OC6-HSL by fusion with maltose-binding protein (MBP). Dissociation constants for 3OC6-HSL from MBP-fusions of WT-, I46F- and I46R-LuxR determined by surface plasmon resonance confirmed the binding affinity. We designed and constructed a novel whole-cell biosensor on the basis of LuxR-I46F in E. coli host cells with a reporting module that expressed green fluorescent protein. The biosensor had high sensitivity in response to the signaling molecule 3OC6-HSL produced by the target bacterial pathogen Yersinia pestis. Our work demonstrates a practical, generalizable framework for the rational design and adjustment of LuxR-family proteins for use in bioengineering and bioelectronics applications. |
| Databáze: | OpenAIRE |
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