Microbial production of aliphatic (S)-epoxyalkanes by using Rhodococcus sp. strain ST-10 styrene monooxygenase expressed in organic-solvent-tolerant Kocuria rhizophila DC2201.

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.

Expression and characterization of styrene monooxygenases of Rhodococcus sp. ST-5 and ST-10 for synthesizing enantiopure (S)-epoxides.

Styrene monooxygenase (StyA, SMOA)- and flavin oxidoreductase (StyB, SMOB)-coding genes of styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10 were successfully expressed in Escherichia coli. Determined amino acid sequences of StyAs and StyBs of ST-5 and ST-10 showed more similarity with those of Pseudomonas than with self-sufficient styrene monooxygenase (StyA2B) of Rhodococcus. Recombinant enzymes were purified from E. coli cells as functional proteins, and their properties were characterized in detail. StyBs (flavin oxidoreductase) of strains ST-5 and ST-10 have similar enzymatic properties to those of Pseudomonas, but StyB of strain ST-10 exhibited higher temperature stability than that of strain ST-5. StyAs of strains ST-5 and ST-10 catalyzed the epoxidation of vinyl side-chain of styrene and its derivatives and produced (S)-epoxides from styrene derivatives and showed high stereoselectivity. Both StyAs showed higher specific activity on halogenated styrene derivatives than on styrene itself. Additionally, the enzymes could catalyze the epoxidation of short-chain 1-alkenes to the corresponding (S)-epoxides. Aromatic compounds including styrene, 3-chlorostyrene, styrene oxide, and benzene exhibited marked inhibition of SMO reaction, although linear 1-alkene showed no inhibition of SMO activity at any concentration.