Robust Characterization of Two Distinct Glutarate Sensing Transcription Factors of Pseudomonas putida l-Lysine Metabolism.

Autor:	Thompson MG; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Department of Plant and Microbial Biology , University of California , Berkeley , California 94720 , United States., Costello Z; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; DOE Agile BioFoundry , Emeryville , California 94608 , United States., Hummel NFC; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Department of Plant Biology , University of California, Davis , Davis , California 95616 , United States., Cruz-Morales P; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Centro de Biotecnologia FEMSA , Instituto Tecnologico y de Estudios Superiores de Monterrey , 64849 Monterrey , Mexico., Blake-Hedges JM; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Department of Chemistry , University of California , Berkeley , California 94720 , United States., Krishna RN; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Department of Chemistry , University of California , Berkeley , California 94720 , United States., Skyrud W; Department of Chemistry , University of California , Berkeley , California 94720 , United States., Pearson AN; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States., Incha MR; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Department of Plant and Microbial Biology , University of California , Berkeley , California 94720 , United States., Shih PM; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Department of Plant Biology , University of California, Davis , Davis , California 95616 , United States., Garcia-Martin H; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; DOE Agile BioFoundry , Emeryville , California 94608 , United States.; BCAM, Basque Center for Applied Mathematics , 48009 Bilbao , Spain., Keasling JD; Joint BioEnergy Institute , 5885 Hollis Street , Emeryville , California 94608 , United States.; Biological Systems & Engineering Division , Lawrence Berkeley National Laboratory , Berkeley , California 94720 , United States.; Joint Program in Bioengineering , University of California , Berkeley , California 94720 , United States.; Department of Chemical and Biomolecular Engineering , University of California , Berkeley , California 94720 , United States.; The Novo Nordisk Foundation Center for Biosustainability , Technical University of Denmark , 2800 Kgs. Lyngby , Denmark.; Center for Synthetic Biochemistry, Institute for Synthetic Biology , Shenzhen Institutes for Advanced Technologies , Shenzhen 518055 , China.
Jazyk:	angličtina
Zdroj:	ACS synthetic biology [ACS Synth Biol] 2019 Oct 18; Vol. 8 (10), pp. 2385-2396. Date of Electronic Publication: 2019 Sep 25.
DOI:	10.1021/acssynbio.9b00255
Abstrakt:	A significant bottleneck in synthetic biology involves screening large genetically encoded libraries for desirable phenotypes such as chemical production. However, transcription factor-based biosensors can be leveraged to screen thousands of genetic designs for optimal chemical production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida . The genomic contexts of csiR homologues were analyzed, and their DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro . CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added, confirming that it acts as a genetic repressor. Both transcription factors and cognate promoters were then cloned into broad host range vectors to create two glutarate biosensors. Their respective sensing performance features were characterized, and more sensitive derivatives of the GcdR biosensor were created by manipulating the expression of the transcription factor. Sensor vectors were then reintroduced into P. putida and evaluated for their ability to respond to glutarate and various lysine metabolites. Additionally, we developed a novel mathematical approach to describe the usable range of detection for genetically encoded biosensors, which may be broadly useful in future efforts to better characterize biosensor performance.
Databáze:	MEDLINE
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