AraC Regulatory Protein Mutants with Altered Effector Specificity

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Abstract
The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to d-arabinose and not in response to its native effector l-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM l-arabinose does not influence the response of the reported mutants to d-arabinose, and the mutants are not induced by other sugars tested (d-xylose, d-fucose, d-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by d-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.