Engineering Terpene Synthases for Increased Productivity

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DESCRIPTION (provided by applicant): Terpenes are the most diverse class of natural products, with 30,000 known members. A number of characterized terpenes form commercially useful products, including the anticancer agent Taxol. Large- scale production of terpenes is difficult because current techniques rely on complex chemical synthesis or inefficient extraction from biological sources. To address the demand for therapeutic terpenes the Keasling laboratory has engineered Escherichia coli to produce amorpha-4,11-diene, the precursor for the anti- malarial compound, artemisinin. However, adequate expression of the terpene synthase, amorphadiene synthase (ADS), has become a major obstacle to increased amorphadiene production. Terpene synthases catalyze the cyclization of polyprenyl diphosphates to form a terpene backbone, which is the first committed step in all terpene biosynthesis. In this proposal, we outline an approach to improve terpene synthase expression in E. coli by increasing both solubility and activity,of the model terpene synthase, ADS, as well as to optimize flux through this enzyme to increase amorphadiene production. Altered ADS enzymes will be analyzed and a model linking terpene synthase sequence to enzyme function will be established.