Functional and Structural Characterization of an IclR Family Transcription Factor for the Development of Dicarboxylic Acid Biosensors
Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and in vivo characterization of TF ligand response as well as detailed molecular structure information. In this work we characterize the PcaR TF belonging to the IclR family. We present in vitro functional analysis of PcaRs ligand profile and construction of genetic circuits for the characterization of PcaR as an in vivo biosensor in the model eukaryote Saccharomyces cerevisiae. We report the crystal structures of PcaR in the apo state and in complex with one of its ligands, succinate, which suggests the mechanism of dicarboxylic acid recognition by this TF. This work provides key structural and functional insights enabling the engineering of PcaR for dicarboxylic acid biosensors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=50 SRC="FIGDIR/small/550818v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@16bcdd2org.highwire.dtl.DTLVardef@95ba9forg.highwire.dtl.DTLVardef@f598borg.highwire.dtl.DTLVardef@1bfe5f2_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPcaR is an IclR family transcription regulator responsive to dicarboxylic acids C_LIO_LIPcaR was established as an in vivo biosensor in yeast C_LIO_LICrystal structure of PcaR in the apo form was solved C_LIO_LICrystal structure with PcaR in complex with succinate was solved C_LIO_LISequence alignments unveil ligand-binding positions in the IclR family C_LI