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

(+)-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.