Directed evolution of VanR biosensor specificity in yeast

Biotechnology Notes, vol. 1, pp. 9–15

Abstract

Allosterically regulated transcription factors (aTFs) based biosensors from prokaryotes have been widely used to screen large gene libraries, stabilize engineered microbes from evolutionary drifting, and for detection of soil pollutants, among many other applications. However, even though aTF-based biosensors have been established as successful tools for bioengineering and remediation, rational engineering of aTF small molecule-specificity have so far not been demonstrated, highlighting the need for a deeper understanding of the sequence-function relationships that govern aTF allostery. Here, by combining directed evolution of a naïve library of VanR, a vanillic acid transcriptional regulator from Caulobacter crescentus in yeast, followed by saturation mutagenesis of selected positions we identify residues required for vanillic acid responsiveness, while at the same time maintaining responsiveness to vanillin. Selected single-position VanR mutants show both complete repression of transcription in the absence of any ligand, complete loss of vanillic acid responsiveness, while still maintaining high derepression in the presence of vanillin. By computational ligand docking analyses we also discuss the structure-function relationship single mutations can have on aTF specificity, an attribute potentially accounting for the wide-spread arise of aTF members belonging to the GntR superfamily of transcriptional regulators.

Authors 9

1. Vasil D’Ambrosio

   Novo Nordisk Foundation · Technical University of Denmark

   Affiliation as printed

   Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark

2. Subrata Pramanik Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany

3. Kati Goroncy
   Affiliation as printed

   SeSaM-Biotech GmbH, Aachen, Germany

4. Tadas Jakočiūnas

   Novo Nordisk Foundation · Technical University of Denmark

   Affiliation as printed

   Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark

5. David Schönauer
   Affiliation as printed

   SeSaM-Biotech GmbH, Aachen, Germany

6. Mehdi D. Davari Aachen Institute of Biotechnology

   RWTH Aachen University

   Affiliation as printed

   Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany

7. Ulrich Schwaneberg Aachen DWI - Leibniz Institute for Interactive Materials Institute of Biotechnology

   DWI – Leibniz Institute for Interactive Materials · RWTH Aachen University

   Affiliation as printed

   DWI − Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, 52074, Aachen, Germany

   Institute of Biotechnology, RWTH Aachen University, Worringer Weg 3, D-52074, Aachen, Germany

8. Jay D. Keasling

   Lawrence Berkeley National Laboratory · Novo Nordisk Foundation · Shenzhen Institutes of Advanced Technology · Joint BioEnergy Institute · University of California, Berkeley · Technical University of Denmark

   Affiliation as printed

   Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

   Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institute of Advanced Technologies, Shenzhen, China

   Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, CA, USA

   Joint BioEnergy Institute, Emeryville, CA, USA

   Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark

9. Michael Krogh Jensen corresponding

   Novo Nordisk Foundation · Technical University of Denmark

   Affiliation as printed

   Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark