A Yeast Three Hybrid Assay for Metabolic Engineering of Tetracycline Derivatives

Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Toward this end, we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Var...