An in vitro platform for the rapid and scalable biocatalysis of complex polyketides

Modular polyketide synthases (PKSs) are enzymatic assembly lines that naturally produce complex, bioactive compounds challenging to obtain through traditional synthesis or fermentation. Early attempts to harness these molecular factories outside the cell indicated they make poor biocatalysts. However, we observe that a refactored pikromycin synthase optimized for in vitro performance operates as rapidly as iterative fatty acid synthases, two orders of magnitude faster than measured for the equivalently-sized erythromycin synthase. The activities of four engineered variants of the pikromycin synthase were also enhanced through relocating histidine tags, swapping enzymatic/docking domains, and tuning polypeptide stoichiometry. To enable the economic conversion of feedstock chemicals malonate and methylmalonate by these and other synthases into scalable quantities of complex polyketides, we developed the Extender unit to PolyKetide (EPK) platform, which incorporates extender unit and NADPH regeneration systems. In a 10-mL, 2-hour, room-temperature reaction, 1 μM of a refactored pikromycin synthase converts malonate and methylmalonte into 2.8 mM (1 mg/mL) narbonolide with a total turnover number (TTON) of 2800. All five stereocomplex products were readily obtained in pure form at the milligram scale and structurally characterized, demonstrating the advantages of accessing complex polyketide through in vitro biocatalysis as well as the potential to access combinatorial polyketide libraries for medicinal chemistry.