We describe the development of biocatalysis for producing optically pure straight-chain (S)-epoxyalkanes using styrene monooxygenase of Rhodococcus sp. strain ST-10 (RhSMO). RhSMO was expressed in the organic solvent-tolerant microorganism Kocuria rhizophila DC2201, and the bioconversion reaction was performed in an organic solvent-water biphasic reaction system. The biocatalytic process enantioselectively converted linear terminal alkenes to their corresponding (S)-epoxyalkanes using glucose and molecular oxygen. When 1-heptene and 6-chloro-1-hexene were used as substrates (400 mM) under optimized conditions, 88.3 mM (S)-1,2-epoxyheptane and 246.5 mM (S)-1,2-epoxy-6-chlorohexane, respectively, accumulated in the organic phase with good enantiomeric excess (ee; 84.2 and 95.5%). The biocatalysis showed broad substrate specificity toward various aliphatic alkenes, including functionalized and unfunctionalized alkenes, with good to excellent ee. Here, we demonstrate that this biocatalytic system is environmentally friendly and useful for producing various enantiopure (S)-epoxyalkanes. Enantiopure epoxides are important compounds for synthesizing chiral materials, including pharmaceuticals, agrochemicals, and fine chemicals (1, 2). Enantioselective epoxidation of prochiral alkenes is a straightforward strategy for producing enantiopure epoxides, and many chemical approaches, such as the Sharpless epoxidation (3, 4), metal-salen catalysts (5-7), and fructose derivatives (8, 9), have been studied to achieve this objective. However, direct enantioselective epoxidation of straight-chain terminal alkenes remains challenging because of the difficulty of controlling the prochiral faces with a simple monosubstituted double bond. Therefore, in organic syntheses, optically pure terminal epoxides are synthesized by the kinetic resolution of racemic epoxides using a cobalt-salen catalyst (10). To address this problem, further improvements of the catalyst for direct enantioselective epoxidation of straight-chain terminal alkenes are necessary.Several enzymes, including cytochrome P450 (11, 12), alkene or toluene monooxygenase (13,14), haloperoxidase (15, 16), and styrene monooxygenase (SMO) (17-19), catalyze asymmetric epoxidation of alkenes to the corresponding epoxide. Compared with the chemical approach, enzymatic epoxidation of alkenes has several advantages, such as high regio-and enantioselectivity and environmental sustainability, although the production levels of the epoxide are relatively low, except in a few cases. Therefore, biocatalytic systems for producing enantiopure epoxides using these enzymes have been extensively studied.SMO is involved in the degradation and catabolism of styrene in some microorganisms (17,20). SMO catalyzes the epoxidation of styrene to (S)-styrene oxide with excellent enantiomeric excess (ee) (Ͼ99%), and it has been well studied as a biocatalyst due to its superior enantioselectivity. SMO consists of two enzymes: flavin oxidoreductase (StyB), which reduces flavin adenine dinucleotide (FAD) ...