(+)‐3,6‐Epoxymaaliane: A Novel Derivative of (+)‐Bicyclogermacrene Oxidation Catalyzed by CYP450 BM3‐139‐3 and Its Variants

Abstract (+)‐Bicyclogermacrene is a sesquiterpene compound found in various plant essential oils and serves as a crucial precursor for multiple biologically active compounds. Many derivatives of (+)‐bicyclogermacrene have been shown to exhibit valuable bioactivities. Cytochrome P450 BM3 from Bacillus megaterium can catalyze a variety of substrates and different types of oxidation reactions, making it become a powerful tool for oxidizing terpenes. In this study, we employed P450 BM3‐139‐3 variant for in vitro enzymatic oxidation of (+)‐bicyclogermacrene, identifying a novel oxidized derivative of (+)‐bicyclogermacrene, named (+)‐3,6‐epoxymaaliane, and an unknown sesquiterpenoid in a ratio of 70 : 30 (by GC peak area). (+)‐3,6‐Epoxymaaliane showed demonstrated antibacterial activities toward Escherichia coli and Staphylococcus aureus . To obtain a better variant of the monooxygenase with a high selectivity to form (+)‐3,6‐epoxymaaliane, we combined alanine scanning with the “Focused Rational Iterative Site‐Specific Mutagenesis” (FRISM) strategy to modify the closest residues within 5 Å radius surrounding the substrate to create a small‐but‐smart library of mutants. Consequently, it gave an optimal variant with 1.6‐fold improvement, in a turnover number (TON) of up to 964 toward (+)‐3,6‐epoxymaaliane production with a higher product selectivity.