(474a) Engineered Variants of AraC Regulatory Protein Responding to Mevalonate and HMGCoA

Novel variants of the AraC regulatory protein were designed to control gene expression in response to mevalonate or HMGCoA, two intermediates in the synthesis of cholesterol and high-value terpenoid compounds. Residue positions in the AraC binding pocket were randomized and a GFP reporter construct was used to screen for AraC mutants with desired regulatory properties in the presence and absence of mevalonate or HMGCoA. This presentation will highlight interesting features and applications of mutants recovered from library screening. A mutant that activates gene expression in the presence of mevalonate was used as a molecular reporter for engineering novel variants of HMGCoA reductase. Synthesis of an HMGCoA reductase inhibitor was also improved using the mevalonate reporter. Another AraC mutant displays atypical regulatory behavior, such that gene activation is inhibited by HMGCoA. This unique reporter can be used to screen for inhibitors of HMGCoA synthesis. Finally, mutational analysis allows us to propose the mechanisms of molecular recognition in these variants and offers insights for future engineering efforts.