Biosynthesis of Fluorinated Aromatic Polyketides Through Directed Evolution of Fused Type III Polyketide Synthase.

Aromatic polyketides have gained significant attention as prominent plant-derived polyphenol molecules in recent years owing to their multi-faceted therapeutic potentials against a variety of diseases. However, the introduction of unique fluorine atoms into the core structure of aromatic polyketides has received limited exploration thus far. In this study, a series of new-to-nature fluorinated aromatic polyketides, including resveratrol, naringenin chalcone, bisnoyangonin, and benzalactone, were obtained from readily available fluoro-tyrosines in E. coli via a multi-enzyme cascade pathway. A series of combinational optimization strategies, including metabolic regulations, double protein fusion (4CL::III-PKS and MatB::III-PKS), and directed evolution of the rate-limiting enzymes, were conducted to improve the production of fluoro-aromatic polyketides, thus enhancing the titers to >200 mg/L from an initial ∼10 mg/L. When both sides of the phenolic hydroxyl group of 4-coumaroyl-CoA contain fluorine atoms, a novel catalytic pathway of STS and CHS in synthesizing methyl ketone structures is unlocked, namely the "proximal fluorine-induced III-PKS catalytic truncation". Further post-functionalizations by enzymatic glycosylation reaction and chemocatalytic Friedel-Crafts-type reaction provided the corresponding fluorinated derivatives of polyphenolic natural products (piceid and arahypin).