SgStyA-Mediated Diastereospecific Epoxidation Of Limonene: Accessing Enantiopure cis- and trans-Limonene 1, 2-Epoxides

Styrene monooxygenases (SMOs) have been widely used in the asymmetric epoxidation of aromatic olefins, but the application of SMOs in the epoxidation of aliphatic alkenes remains limited. Herein, we present an exploration of the SMO-catalyzed epoxidation of terpenes. The SMO from Streptomyces gardneri (SgStyA) was demonstrated to catalyze the asymmetric epoxidation of (R)-(+)-limonene with high regioselectivity and good diastereoselectivity, yielding trans-(+)-limonene-1, 2-epoxide with a diastereomeric ratio of 21:79. Structure-guided protein engineering identified residues 50, 194, and 209 as key determinants of SgStyA diastereoselectivity by governing substrate-binding orientation. The variants SgStyA-M1 (F50L/V209I/G97A) and SgStyA-M2 (F50M/V209I/G97A), harboring a small flexible residue at position 50, exhibited cis-selectivity. In contrast, SgStyA-M3 (F194M/F50W), containing a bulky residue at position 50 and smaller residues at 194 and 209, favored trans-selectivity. All three variants demonstrated highly diastereoselective epoxidation of (R)- and (S)-limonene, enabling the preparative-scale synthesis of enantiopure epoxides including cis-(+)-, trans-(+)-, trans-(−)-, and cis-(−)-limonene-1,2-epoxide. These findings highlight the significant potential of SMO-catalyzed epoxidation of terpene as a green process to high-value enantiopure epoxides from renewable feedstocks, expanding the biocatalytic toolbox for stereoselective epoxidations.