Evolution-guided engineering of small-molecule biosensors
| Autor: | Stefan Kol, Tim Snoek, Jay D. Keasling, Evan K. Chaberski, Michael Krogh Jensen, Francesca Ambri, Bo Pang, Ditte Hededam Welner, Sara Petersen Bjørn, Jesus F. Barajas |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Models
Molecular Secondary Computer science Biosensing Techniques Ligands Protein Structure Secondary Synthetic biology Genes Reporter Models 0303 health sciences 030302 biochemistry & molecular biology Biological Sciences Directed evolution Small molecule Sorbic Acid DNA-Binding Proteins Generic Health Relevance Methods Online Eukaryote Genetic Engineering Biotechnology Protein Structure Green Fluorescent Proteins Protein domain Saccharomyces cerevisiae Allosteric regulation Bioengineering Computational biology Biology 03 medical and health sciences Protein Domains Information and Computing Sciences Escherichia coli Genetics Reporter Transcription factor Gene Library 030304 developmental biology Cellular metabolism 030306 microbiology Ligand Molecular Prokaryote DNA biology.organism_classification Yeast Evolvability Genes Mutagenesis Directed Molecular Evolution Biosensor Function (biology) Environmental Sciences Transcription Factors Developmental Biology |
| Zdroj: | Snoek, T, Chaberski, E K, Ambri, F, Kol, S, Bjørn, S P, Pang, B, Barajas, J F, Welner, D H, Jensen, M K & Keasling, J D 2020, ' Evolution-guided engineering of small-molecule biosensors ', Nucleic Acids Research, vol. 48, no. 1, e3 . https://doi.org/10.1093/nar/gkz954 Nucleic Acids Research Nucleic acids research, vol 48, iss 1 |
| Popis: | Allosteric transcription factors (aTFs) have proven widely applicable for biotechnology and synthetic biology as ligand-specific biosensors enabling real-time monitoring, selection and regulation of cellular metabolism. However, both the biosensor specificity and the correlation between ligand concentration and biosensor output signal, also known as the transfer function, often needs to be optimized before meeting application needs. Here, we present a versatile and high-throughput method to evolve and functionalize prokaryotic aTF specificity and transfer functions in a eukaryote chassis, namely baker’s yeastSaccharomyces cerevisiae. From a single round of directed evolution of the effector-binding domain (EBD) coupled with various toggled selection regimes, we robustly select aTF variants of thecis, cis-muconic acid-inducible transcription factor BenM evolved for change in ligand specificity, increased dynamic output range, shifts in operational range, and a complete inversion of function from activation to repression. Importantly, by targeting only the EBD, the evolved biosensors display DNA-binding affinities similar to BenM, and are functional when ported back into a non-native prokaryote chassis. The developed platform technology thus leverages aTF evolvability for the development of new host-agnostic biosensors with user-defined small-molecule specificities and transfer functions. |
| Databáze: | OpenAIRE |
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