Epoxidation of perillyl alcohol by engineered bacterial cytochrome P450 BM3

Abstract

Perillyl alcohol (POH) is a secondary metabolite of plants. POH and its derivatives are known to be effective as an anticancer treatment. In this study, oxidative derivatives of POH, which are difficult to synthesize chemically, were synthesized using the engineered bacterial cytochrome P450 BM3 (CYP102A1) as a biocatalyst. The activity of wild-type (WT) CYP102A1 and 29 engineered enzymes toward POH was screened using a high-performance liquid chromatography. They produced one major product. Among them, the engineered CYP102A1 M601 mutant with seven mutations (R47L/F81I/F87V/E143G/L150F/L188Q/E267V) showed the highest conversion, 6.4-fold higher than the WT. Structure modeling using AlphFold2 and PyMoL suggests that mutations near the water channel may be responsible for the increased catalytic activity of the M601 mutant. The major product was identified as a POH-8,9-epoxide by gas chromatography-mass spectrometry and nuclear magnetic resonance analysis. The optimal temperature and pH for the product formation were 35 °C and pH 7.4, respectively. The k_cat and K_m of M601 were 540 min⁻¹ and 2.77 mM, respectively. To improve POH-8,9-epoxide production, substrate concentration and reaction time were optimized. The optimal condition for POH-8,9-epoxide production by M601 was 5.0 mM POH, pH 7.4, 35 ℃, and 6 h reaction, which produced the highest concentration of 1.72 mM. Therefore, the biosynthesis of POH-8,9-epoxide using M601 as a biocatalyst is suggested to be an efficient and sustainable synthetic process that can be applied to chemical and pharmaceutical industries.