The development and industrial production of antibiotics involves an intimate interplay between biological and synthetic chemistries. Biological systems have been selected throughout evolution to generate highly bioactive products through pathways with stereoselective catalytic activity and unmatched atom economy. However, natural products typically have poor pharmacological properties and synthetic chemistry is employed to modify them into useful drugs. These synthetic steps frequently feature low yields, involve the use of toxic reagents, and contribute substantially to the overall cost of drug development. Thus, the evolution of enzymes that circumvent these synthetic steps by operating on unnatural substrate analogues [and perform new biochemical transformations] is important for the efficient conversion of potent natural products into clinically-useful drugs. This proposal outlines a strategy to subvert the natural biosynthetic pathway of nosiheptide, a thiopeptide antibiotic, to generate nosiheptide analogues with improved activity and solubility. We have identified the radical SAM enzyme NosL, as an ideal target for this objective. Radical SAM enzymes perform chemically challenging reactions, such as the radical fragmentation-recombination reaction of NosL, in many important biosynthetic pathways. Directed evolution of radical SAM enzymes has not been performed and [we will use this opportunity to uncover efficient strategies for tuning their reactivity].
Our specific aims are: [(1) To expand the substrte profile of NosL through directed evolution; (2) Use NosL to establish efficient techniques for uncovering new RS chemistry and]; (3) Introduce engineered nosL genes into the native producer to ferment regiospecifically modified nosiheptides analogues. These antibiotics may then be further modified through cross-coupling chemistry to access pharmacologically-promising non-natural products. All precursors may be produced on preparative-scale with the promiscuous enzyme tryptophan synthase, synthesized with established techniques, or purchased from commercial vendors.
Naturally occurring drugs may be improved upon by engineering their underlying biosynthetic pathways. The proposed research will establish the first directed evolution of an important class of enzymes. These catalysts will redirect the pathway for production the antibiotic nosiheptide to generate new and more effect drugs.
