Development of a Biosensor Platform for Phenolic Compounds Using a Transition Ligand Strategy

ACS Synthetic Biology, vol. 10, pp. 2002–2014

Abstract

High Resolution Image Download MS PowerPoint Slide The time-consuming and laborious characterization of protein or microbial strain designs limits the development of high-performance biocatalysts for biotechnological applications. Here, transcriptional biosensors emerged as valuable tools as they allow for rapid characterization of several thousand variants within a very short time. However, for many molecules of interest, no specific transcriptional regulator determining a biosensor’s specificity is available. We present an approach for rapidly engineering biosensor specificities using a semirational transition ligand approach combined with fluorescence-activated cell sorting. In this two-step approach, a biosensor is first evolved toward a more relaxed-ligand specificity before using the resulting variant as the starting point in a second round of directed evolution toward high specificity for several chemically different ligands. By following this strategy, highly specific biosensors for 4-hydroxybenzoic acid, p -coumaric acid, 5-bromoferulic acid, and 6-methyl salicylic acid were developed, starting from a biosensor for the intracellular detection of trans -cinnamic acid.

Authors 4

1. Lion Konstantin Flachbart

   Forschungszentrum Jülich

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany

2. Christoph G. W. Gertzen

   Forschungszentrum Jülich · John von Neumann Institute for Computing · Jülich Supercomputing Centre · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Center for Structural Studies (CSS), Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany

   Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany

   John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC) and Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany

3. Holger Gohlke

   Forschungszentrum Jülich · John von Neumann Institute for Computing · Jülich Supercomputing Centre · Heinrich Heine University Düsseldorf

   Affiliation as printed

   Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany

   John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC) and Institute of Biological Information Processing (IBI-7: Structural Biochemistry), Forschungszentrum Jülich GmbH, D-52425 Jülich, Germany

4. Jan Marienhagen corresponding Aachen Institute of Biotechnology

   Forschungszentrum Jülich · RWTH Aachen University

   Affiliation as printed

   Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, D-52425 Jülich, Germany

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