Engineered regulatory proteins enable customized genetic selections and permit targeted gene transcription for applications in metabolic engineering, biosensing and genetic circuit design. Use of regulatory proteins for reporting or controlling metabolic pathways in response to specific metabolites requires a selective protein-effector interaction that is not influenced by similar compounds, such as metabolic precursors or undesired co-products. The AraC dual regulatory protein naturally regulates the ara operon in response to L-arabinose in E. coli. We are engineering AraC to respond to a variety of non-native ligands. Structure analysis of the AraC binding pocket is used to suggest mutant library design strategies. Simultaneous saturation mutagenesis at different combinations of binding pocket residues (yielding ~10⁶-10⁷ variants per library) followed by dual screening yields mutants selectively inducible by ligands of interest and not by chosen decoy compounds. This approach has proven successful for a variety of new ligands, including sugars and metabolites. Experimental and computational analyses of the mutants reveal insights into the AraC induction response and the roles of various residues in molecular recognition and/or switching from a repressing to an activating conformation. These studies include comparisons between ligand binding affinity in vitro and half-maximal induction response in vivo. In general, N-terminal arm mutations reduce the arm's ability to maintain a transcriptional repression conformation (by interacting with the C-terminus), thereby lowering the ligand binding affinity required for transcriptional activation. The consequence of this relaxed switch is often an increase in constitutive expression. Compensatory mutations within the binding pocket can serve to increase ligand binding affinity and reduce constitutivity, although at the cost of the strength of the induction response. Our studies suggest the AraC N-terminal arm plays little role in ligand binding affinity. Preservation of native arm-domain interactions during mutagenesis demands a tighter binding pocket-ligand interaction for transcriptional activation.

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