Switching the Ligand Specificity of the Biosensor XylS from meta to para-Toluic Acid through Directed Evolution Exploiting a Dual Selection System

2019 ◽  
Vol 8 (12) ◽  
pp. 2679-2689
Author(s):  
Yuki Ogawa ◽  
Yohei Katsuyama ◽  
Kento Ueno ◽  
Yasuo Ohnishi
Keyword(s):  
Directed Evolution ◽  
Ligand Specificity ◽  
Selection System

scholarly journals PROFICS: A bacterial selection system for directed evolution of proteases

2021 ◽  
pp. 101095
Author(s):  
Christina Kröß ◽  
Petra Engele ◽  
Bernhard Sprenger ◽  
Andreas Fischer ◽  
Nico Lingg ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Selection System ◽  
Bacterial Selection

Directed Evolution of The PobR Allosteric Transcription Factor To Generate A Biosensor For 4-Hydroxymandelic Acid

2021 ◽  
Author(s):  
YaoYao Liang ◽  
Juan Luo ◽  
Chenhao Yang ◽  
Shuning Guo ◽  
Bowen Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Directed Evolution ◽  
Missense Mutation ◽  
Hydroxybenzoic Acid ◽  
Ligand Specificity ◽  
Mutant Library

Abstract 4-Hydroxymandelic 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.

scholarly journals The directed evolution of ligand specificity in a GPCR and the unequal contributions of efficacy and affinity

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Raphaël B. Di Roberto ◽  
Belinda Chang ◽  
Sergio G. Peisajovich
Keyword(s):  
Directed Evolution ◽  
Ligand Specificity

scholarly journals An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Parinthon Nearmnala ◽  
Manutsawee Thanaburakorn ◽  
Watanalai Panbangred ◽  
Pimchai Chaiyen ◽  
Narupat Hongdilokkul
Keyword(s):  
Directed Evolution ◽  
Selection Pressure ◽  
Catalytic Efficiency ◽  
Selection System ◽  
In Vivo Selection ◽  
Highly Efficient ◽  
Highly Active ◽  
Wide Range ◽  
Regulated Gene Expression

AbstractIn vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.

scholarly journals Directed evolution of a transcription factor PbrR to improve lead selectivity and reduce zinc interference through dual selection

2020 ◽  
Author(s):  
Xiaoqiang Jia ◽  
Yubing Ma ◽  
Rongrong Bu ◽  
Tingting Zhao ◽  
Kang Wu
Keyword(s):  
Transcription Factor ◽  
Directed Evolution ◽  
Metal Binding ◽  
Binding Specificity ◽  
Selection Marker ◽  
Selection System ◽  
Lead And Cadmium ◽  
Negative Selection Marker ◽  
Binding Residue ◽  
Ampicillin Resistance Gene

Abstract Directed evolution has been proven as a powerful tool for developing proteins and strains with novel or enhanced features. In this study, a dual selection system was designed to tune the binding specificity of a transcription factor to a particular ligand with the ampicillin resistance gene amp (ON selection) as the positive selection marker and the levansucrase gene sacB (OFF selection) as the negative selection marker. It was applied to the lead responsive transcription factor PbrR in a whole-cell lead biosensor previously constructed in our lab. After multiple rounds of ON-OFF selection, two mutants with higher specificity for lead were selected. Structural analysis revealed that the mutation C134 located on the metal-binding loop at the C-terminal of PbrR is likely associated with the enhanced binding to both lead and cadmium. The double mutations D64A and L68S close to the metal-binding residue C79 may lead to the reduced binding specificity toward zinc ions. This dual selection system can be applied to engineer the specificity of other transcription factors and provide fine-tuned tools to synthetic biology.

Directed Evolution of Orthogonal Ligand Specificity in a Single Scaffold

2009 ◽  
Vol 48 (42) ◽  
pp. 7783-7786 ◽  
Author(s):  
Michael J. McLachlan ◽  
Karuppiah Chockalingam ◽  
Ka Chun Lai ◽  
Huimin Zhao
Keyword(s):  
Directed Evolution ◽  
Ligand Specificity

scholarly journals Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Leopoldo F. M. Machado ◽  
Andrew Currin ◽  
Neil Dixon
Keyword(s):  
Transcription Factor ◽  
Directed Evolution ◽  
Aromatic Aldehydes ◽  
Ligand Specificity ◽  
Phenotypic Analysis ◽  
Potential Applications ◽  
Substituted Benzoic Acids

Abstract Background Transcription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal. Results Here, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance. Conclusions This is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.

scholarly journals A generic selection system for improved expression and thermostability of G protein-coupled receptors by directed evolution

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Christoph Klenk ◽  
Janosch Ehrenmann ◽  
Marco Schütz ◽  
Andreas Plückthun
Keyword(s):  
Directed Evolution ◽  
G Protein ◽  
G Protein Coupled Receptors ◽  
Selection System ◽  
G Protein Coupled

Directed Evolution of Orthogonal Ligand Specificity in a Single Scaffold

2009 ◽  
Vol 121 (42) ◽  
pp. 7923-7926
Author(s):  
Michael J. McLachlan ◽  
Karuppiah Chockalingam ◽  
Ka Chun Lai ◽  
Huimin Zhao
Keyword(s):  
Directed Evolution ◽  
Ligand Specificity

Pilot selection system development.

1978 ◽  
Author(s):  
David R. Hunter ◽  
Nancy A. Thompson
Keyword(s):  
System Development ◽  
Selection System ◽  
Pilot Selection
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