Skeletal editing via multi-step engineering of a modular polyketide synthase.

Assembly line biosynthesis creates numerous structurally diverse natural products using a common modular synthetic strategy. The collinearity between the architectures of modular polyketide synthases (PKS) and the structures of their polyketide products would seem to render these biosynthetic machineries excellent platforms for designer biosynthesis, yet reliable strategies to reprogram these assembly lines without diminishing their activities have not been identified. Here, as a best practice for PKS engineering, we demonstrate the reprogramming of the mediomycin PKS without significant loss of productivity. Using in vitro CRISPR/Cas9 gene editing followed by heterologous expression, we reconstruct an inaccessible drug lead of the fibrinogen receptor, tetrafibricin, at 82 ± 3 mg/L yield, retaining 26% productivity after five-step module editing using an evolution-supported cut site, downstream of the acyltransferase domain. A macrocyclic aminopolyol is also accessed through thioesterase swapping. These results pave the way toward the rational reprogramming of PKSs to access desired complex organic molecules.