The malonyl/acetyl-transferase from murine fatty acid synthase is a promiscuous engineering tool for editing polyketide scaffolds.

Modular polyketide synthases (PKSs) play a vital role in the biosynthesis of complex natural products with pharmaceutically relevant properties. Their modular architecture makes them an attractive target for engineering to produce platform chemicals and drugs. In this study, we demonstrate that the promiscuous malonyl/acetyl-transferase domain (MAT) from murine fatty acid synthase serves as a highly versatile tool for the production of polyketide analogs. We evaluate the relevance of the MAT domain using three modular PKSs; the short trimodular venemycin synthase (VEMS), as well as modules of the PKSs deoxyerythronolide B synthase (DEBS) and pikromycin synthase (PIKS) responsible for the production of the antibiotic precursors erythromycin and pikromycin. To assess the performance of the MAT-swapped PKSs, we analyze the protein quality and run engineered polyketide syntheses in vitro. Our experiments include the chemoenzymatic synthesis of fluorinated macrolactones. Our study showcases MAT-based reprogramming of polyketide biosynthesis as a facile option for the regioselective editing of substituents decorating the polyketide scaffold.