Refactoring pikromycin synthase for the modular biosynthesis of macrolide antibiotics in E. coli

Abstract

While engineering modular polyketide synthases (PKSs) using the recently updated module boundary has yielded libraries of triketide–pentaketides, this strategy has not yet been applied to the combinatorial biosynthesis of macrolactones or macrolide antibiotics. Here we developed a two-plasmid system for the construction and expression of PKSs and used it to obtain a refactored pikromycin PKS in Escherichia coli that produces 49 mg of narbonolide per litre of culture. The replacement, insertion, deletion and mutagenesis of modules enabled access to hexaketide, heptaketide and octaketide derivatives. Supplying enzymes for desosamine biosynthesis and transfer enabled production of narbomycin, pikromycin, YC-17, methymycin and five derivatives thereof. By using native docking domains, knocking out pathways competing with desosamine biosynthesis and supplying the editing thioesterase PikAV, the titre of narbomycin was boosted 58-fold to 37 mg l−1. The second and third modules of the refactored pikromycin synthase were swapped with others to yield three new narbomycin analogues, investigate structure–activity relationships and demonstrate how libraries of macrolide antibiotics can be readily accessed.