(383a) Combinatorial Biosynthesis Of Targeted Tetracycline Polyketides Through Protein Engineering

Combinatorial biosynthesis is an effective way to produce valuable unnatural natural products with improved biological activities. However the vast potential of combinatorial biosynthesis is far from being realized due to the lack of general programming rules for recombining enzymes from different sources. We will develop protein engineering tools to understand these intrinsic limitations and engineer natural product biosynthesis. Three interconnected components in the metabolite-responsive regulation circuitry will be used to allow host cell behavior to be dictated by the presence of a specific metabolite, which is biosynthesized by the engineered pathway. The first component is a reconstituted pathway in an organism that is genetically amendable to the directed evolution techniques. The second component is a regulatory cascade that responds to a target metabolite. The third component of the design is a combinatorial library of genes encoding mutants of the target enzyme. This design will enable powerful selection of desired enzyme that can function with a specific pathway and its product. We will use the tetracycline-TetR pair as the proof-of-concept system to study the robustness of the cascade.