Cell factories converting bio-based precursors to chemicals present an attractive avenue to a 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 attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an l-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate l-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.

Background:The dynamics of coupling of ATP hydrolysis with transport in ATP-binding cassette transporters is not well understood. Results: Characterization of ATP hydrolysis in the MsbA nucleotide-binding domain by time-resolved FTIR spectroscopy revealed two rate constants for ATP binding with dimerization and hydrolysis. Conclusion: ATP hydrolysis is rate-limiting. Significance: The identification of the IR fingerprints of the motor domain will facilitate real-time analysis of the full-length MsbA transport cycle.

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