Plug-and-play engineering of modular polyketide synthases

Modular polyketide synthases (PKSs) are multidomain, assembly line enzymes that biosynthesize complex antibiotics such as erythromycin and rapamycin. The modular characteristic of PKSs makes them an ideal platform for the custom production of designer polyketides by combinatorial biosynthesis. However, engineered hybrid PKS pathways often exhibit severe loss of enzyme activity, and a general principle for PKS reprogramming has not been established. Here we present a widely applicable strategy for designing hybrid PKSs. We reveal that two conserved motifs are robust cut sites to connect modules from different PKS pathways and demonstrate the custom production of polyketides with different starter units, extender units and variable reducing states. Furthermore, we expand the applicability of these cut sites to construct hybrid pathways involving cis-AT PKS, trans-AT PKS and even nonribosomal peptide synthetase. Collectively, our findings enable plug-and-play reprogramming of modular PKSs and facilitate the application of assembly line enzymes toward the bioproduction of designer molecules.