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

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. We developed a 2-plasmid system for the construction and expression of PKSs and employed it to obtain a refactored pikromycin synthase in E. coli that produces 85 mg of narbonolide per liter 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 6 derivatives thereof. Knocking out pathways competing with desosamine biosynthesis and supplying the editing thioesterase PikAV boosted the titer of narbomycin 55-fold to 37 mgL^-1. The replacement of the 3rd pikromycin module with its 5th yielded a new macrolide antibiotic and demonstrates how libraries of macrolide antibiotics can be readily accessed.