Engineering and application of a biosensor with focused ligand specificity

Microbial cell factories efficiently converting bio-based precursors to chemicals represent an attractive avenue to a more sustainable economy. Yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low throughput endeavor. For this reason, transcriptional biosensors attracted attention as they allow screening of vast libraries in combination with fluorescence-activated cell sorting. However, a broad ligand specificity of many transcriptional regulators often prohibits the development of such ultra-high-throughput screens. We here solved the structure of the transcriptional regulator LysG of Corynebacterium glutamicum detecting all three basic amino acids. Semi-rational engineering of LysG using a FACS-based screening/counterscreening approach yielded a new biosensor bearing a regulator insensitive to l-lysine. Molecular dynamics simulations uncovered the underlying structure-function relationships of a crucial amino acid substitution. As proof of principle, we applied the new biosensor and isolated several l-histidine producing strains by FACS.