Chemoenzymatic Synthesis of Norisoprenoid Aroma Compounds via C–H Activation by Engineered P450 BM3

Norisoprenoid compounds such as the damascones, damascenones, and megastigmatrienones are widely used in the flavor and fragrance industry. Their low natural abundance and the limitations of traditional synthetic routes, such as high energy demands, use of toxic reagents, and challenges in isomeric selectivity, hinder production under mild conditions. Here, we report chemoenzymatic synthesis routes to these compounds using engineered P450BM3 variants for late-stage C–H activation. Screening of α- and β-damascone with a panel of 96 P450BM3 variants revealed high conversion rates and regioselectivities for the intermediates for acid-catalyzed dehydration to form γ- and β-damascenone, respectively. Megastigmatriene oxidation did not give tabanone due to rearrangement to β-ionol, but alternative routes via α-ionol and α-ionone oxidation yielded a mixture of tabanone isomers. Beyond allylic oxidation, the enzyme collection also oxidized these norisoprenoids at the less reactive sp3 aliphatic positions, expanding the diversity of accessible products. Scalability of enzymatic oxidation was demonstrated by the high titer (7.3 g/L), conversion (95%), and total turnover number (9500) for β-damascone oxidation. The findings demonstrate the power of chemoenzymatic strategies in accessing complex norisoprenoids in fewer steps than chemical synthesis routes and lay the groundwork for scalable biotechnological production processes.