CAREER: Engineering Regulatory Protein Effector Specificity to Facilitate Combinatorial Library Analyses

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0644678CirinoThis research project addresses the need for improved technologies for engineering custom-designed enzymes and microbial factories by demonstrating a general approach to developing genetic selections or fluorescence-activated cell sorting (FACS) based screens to facilitate the rapid isolation of improved enzyme or metabolic pathway variants from diverse combinatorial libraries. Two-step approach was proposed, where a dual selection system is first used to engineer a regulatory protein such that the product/metabolite of interest inimitably acts as inducer of transcriptional activation. In the second step, the dual selection system incorporating the modified regulator serves as a highly specific reporter, correlating product formation with a selectable cellular phenotype. This approach will be demonstrated using the AraC regulator and its cognate promoter ("PBAD") as a model system. The proposed research will demonstrate the integration of protein engineering and directed evolution with metabolic engineering, yielding novel or improved synthesis of bioproducts. The broader impacts of this plan lie in the development of a technology generally enabling advances in biocatalysis. Importantly, the demonstrated method is readily extrapolated to microorganisms other than E. coli. The integrated education plan will strengthen the biochemical engineering curriculum and provide invaluable research experiences to students, preparing them for careers in biotechnology. Planned outreach efforts will promote enthusiasm in science and engineering to a broader community by providing research opportunities to underrepresented and educationally or economically disadvantaged groups.