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

Kourist, R.; Nigl, A.; Delsoglio, V.; Grgic, M.; Malihan-Yap, L.; Myrtollari, K.; Spasic, J.; Winkler, M.; Oberdorfer, G.; Taden, A.; Anic, I.

doi:10.1101/2024.07.04.601532

bioRxiv · 10.1101/2024.07.04.601532

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

Kourist, R.·Nigl, A.·Delsoglio, V.·Grgic, M.·Malihan-Yap, L.·Myrtollari, K.·Spasic, J.·Winkler, M.·Oberdorfer, G.·Taden, A.·Anic, I.

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Abstract

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.

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Kourist, R., Nigl, A., Delsoglio, V., Grgic, M., Malihan-Yap, L., Myrtollari, K., Spasic, J., Winkler, M., Oberdorfer, G., Taden, A., Anic, I.. 2024-07-08. Engineering of Transmembrane Alkane Monooxygenases to Improve a Key Reaction Step in the Synthesis of Polymer Precursor Tulipalin A. https://doi.org/10.1101/2024.07.04.601532

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