Engineering and Application of Prokaryotic Transcriptional Regulators As Biosensors in Yeast

Bio-based production of chemicals and fuels is an attractive avenue in order to reduce dependence on fossil fuels. However, the current efficiency of genome engineering methods and parts prospecting allow for unprecedented genotype diversity that vastly outstrips our ability to screen for best cell performance. Here we describe the systematic characterization of parameters important for the successful engineering of biosensors for high-throughput screening of yeast cell factories.  Our multi-parametric engineering of a ligand-binding prokaryotic transcription factor for aromatic compound catabolism allowed us to develop a biosensor for dicarboxylic acid accumulation in yeast cell factories. Using the optimal design of the dicarboxylic acid biosensor, we tested a number of other candidate biosensors derived from ligand-binding prokaryotic transcriptional regulators, and here report successful examples of their direct transplantation into yeast cell factories. Most importantly, the biosensors engineered and characterized in our laboratory are applicable for in vivo screening and selection of optimal cell factory performance in a high-throughput manner within relevant operational ranges. Furthermore, omics data analysis suggests that they have no off-target imprint on the native transcriptome. Acknowledging the vast repertoire of ligand-binding transcriptional regulators in prokaryotes, these findings should be of interest to the metabolic engineering community in order to develop future high-throughput screening and selection tools of cell factory libraries targeting bio-based production of a wide range of both fine and bulk chemicals.