Design of a Multienzyme Derived from Mouse Fatty Acid Synthase for the Compartmentalized Production of 2‐Pyrone Polyketides

Abstract

Abstract Compartmentalizing biosynthetic pathways is a key objective in protein engineering, particularly in synthetic biology and metabolic engineering. It can improve catalytic efficiency, stabilize reactive intermediates, reduce by‐product formation, and, beyond these advantages, enable synthetic complexity by coordinating multistep pathways. In this study, we established a chemoenzymatic platform for producing 2‐pyrones—specifically styrylpyrones and hispidin—within a multienzyme based on a non‐reducing (nr) variant of the murine fatty acid synthase (FAS), which naturally produces palmitic acid. By introducing two amino acid substitutions in the ketosynthase (KS) domain, we enhanced the nrFAS‐mediated synthesis of styrylpyrones from non‐native substrates, including halogenated derivatives. The engineered enzyme exhibited a 66‐fold increase in activity compared to the non‐mutated nrFAS, surpassing the styrylpyrone synthase of the kavalactone pathway in Piper methysticum . Additionally, we integrated a 4‐coumarate ligase (4CL1) loading module into the compartment using the SpyTag/SpyCatcher system, enabling the activation and direct transfer of cinnamic acid derivatives to the nrFAS. The resulting styrylpyrones are direct precursors of pharmaceutically active kavalactones, while hispidin serves as the precursor of fungal bioluminescence.