Engineering of Transmembrane Alkane Monooxygenases to Improve a Key Reaction Step in the Synthesis of Polymer Precursor Tulipalin A

The α‐methylene‐γ‐butyrolactone tulipalin A, a defense compound found in tulips, can polymerize via addition at the vinyl group or via ring‐opening polymerization, making it a highly promising monomer for bio‐based polymers. Since the biosynthesis of tulipalin A in plants remains elusive, we propose an alternative pathway for its synthesis starting from the terpenoid intermediate isoprenyl acetate. While fungal unspecific peroxygenases showed a preference for the unwanted epoxidation of its exo‐olefin group, bacterial alkane monooxygenases were selective for terminal hydroxylation. By combining protein engineering based on de novo structure prediction of the membrane enzymes with cell engineering, the specific activity was increased 6‐fold to 1.83 U gcdw‐1. Oxidation of the formed allylic alcohol by a three‐enzyme cascade and subsequent lactonization yielded tulipalin A. Our results demonstrate the feasibility of producing the polymer precursor tulipalin A from terpenoid intermediates and provide a solid foundation for future metabolic engineering endeavors.