Orthogonal Control of Refactored Terpenoid Operon by Engineered T7 RNAP and Zinc Finger Array Transcription Factors

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Brendan Hussey, David McMillen

University of Toronto, Canada

Terpenoids make up the largest class of natural organic compounds and include medically and commercially important members such as artemisinin and isoprene, respectively. As such, the terpenoid pathway has been the subject of much investigation for improving efficiency and increasing yields. Refactoring is a recently applied technique with potential for further enhancing metabolic engineering approaches through optimization of the gene cluster and its regulation. This refactoring approach is being applied to the terpenoid pathway and its precursor MEP pathway to optimize beta-carotene production in E. coli. The refactored operon is regulated by novel transcriptional machinery composed of a modified T7 RNAP and zinc finger array (ZFA) transcription factors. Development of this system will allow for the induction of transcription at any location definable by zinc finger arrays, essentially creating a promoter at this location. In addition, the possibility of regulating protein-protein interactions between the modified T7 and the ZFAs, can allow for a highly flexible system for inducing and tuning transcription. Preliminary experimental results will be presented.