Engineering of Transmembrane Alkane Monooxygenases to Improve a Key Reaction Step in the Synthesis of Polymer Precursor Tulipalin A
The -methylene-{gamma}-butyrolactone tulipalin A, naturally found in tulips can polymerize via addition at the vinyl group or via ring-opening polymerization, making it a highly promising monomer for biobased polymers. As tulipalin A biosynthesis in plants remains elusive, we propose a pathway for its synthesis starting from the metabolic intermediate isoprenol. For this, terminal hydroxylation of the -methylene substrate isoprenyl acetate is a decisive step. While a panel of fungal unspecific peroxygenases showed a preference for the undesired epoxidation of the exo-olefin group, bacterial alkane monooxygenases were specific for terminal hydroxylation. A combination of protein engineering based on de novo structure prediction of the membrane enzymes with cell engineering allowed to increase the specific activity by 6-fold to 1.83 U gcdw -1, unlocking this reaction for the fermentative production of tulipalin A from renewable resources.