In vivo and in vitro hydroxylation of cineole and camphor by cytochromes P450CYP101A1, CYP101B1 and N242A CYP176A1

• 1,8-cineole, (1R) and (1S)-camphor oxidation by CYP101A1, CYP101B1 and N242A-CYP176A1.
• Suite of seven hydroxycamphors and seven hydroxycineoles produced and characterised.
• Whole cell CYP101B1 system can generate between 300 and 600 mg/L of oxidation products.
• Metabolites useful for mechanistic studies and structure elucidation.

Cytochromes P450 (P450s) are valuable enzymes that can generate a range of useful compounds via biocatalytic oxidations that complement traditional synthetic chemistry. In this study three bacterial P450s, P450cam (CYP101A1), CYP101B1 and the mutant N242A-P450cin (N242A-CYP176A1), were used to produce a range of products from the oxidation of the monoterpenes (1R)- and (1S)-camphor and 1,8-cineole. We demonstrate that both in vitro and in vivo catalytic turnover with these P450s can produce a complement of up to seven hydroxycamphors and seven hydroxycineoles, in addition to compounds produced from further oxidation. The CYP101B1 whole cell catalytic system was found to produce 300–600 mg/L of culture of oxidation products that could be easily separated chromatographically. The CYP101B1 in vitro oxidation of 1,8-cineole primarily produced (1S)-5α-hydroxycineole, which was 78% of the total product formed. However, the amount of (1S)-5α-hydroxycineole was reduced to 42% of the total products when isolated from the CYP101B1 whole cell system. (1S)-6α-Hydroxycineole (96% ee) could be isolated from a whole cell catalytic turnover of 1,8-cineole by N242A-P450cin in a yield of 46 mg/L (98% of the total product). However, the amount of product isolated ((1R)-5-endo-hydroxycamphor, 75% of the total products) from the whole cell catalytic oxidation of (1R)-camphor with N242A-P450cin was much lower (6 mg/L) due to the inefficient use of reducing equivalents (3.5 ± 0.5%) for substrate oxidation. These compounds will assist in the identification of specific structures in mechanistic investigations and structure elucidation, but further optimisation is required to generate larger quantities for synthetic applications.

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