Biocatalytic conversion of the natural compound bergamottin into novel bioactive epoxy-derivatives using engineered cytochrome P450 BM3

The bioconversion of natural products by cytochrome P450 enzymes can represent a sustainable alternative to classical chemical synthesis. In this study, we applied a colorimetric assay based on styrene oxidation, to select natural compounds as candidate substrates for cytochrome P450-mediated biocatalysis. One of the tested molecules, bergamottin, was identified as a new substrate for cytochrome P450 BM3 and its D251G/Q307H double mutant, that demonstrated enhanced catalytic efficiency, achieving 87 % conversion within one hour, significantly outperforming the wild-type enzyme. We observed also a 3-fold increase in the total turnover number when comparing engineered enzyme with the wild-type protein. The enzymatic reaction yielded three bergamottin epoxy-derivatives. They were isolated and identified by LC-MS as 2′,3′-epoxybergamottin, 6′,7′-epoxybergamottin, and 2′,3′-6′,7′-diepoxybergamottin. Notably, it is the first time that a bacterial P450 is reported to catalyze epoxidation reaction on bergamottin. Biological assays demonstrated that epoxidation markedly altered bergamottin bioactivity. In particular, 2′,3′-6′,7′-diepoxybergamottin exhibited novel antimicrobial properties and 2′,3′-epoxybergamottin had a beneficial effect on fibroblast viability. These findings highlight the potential of engineered cytochrome P450 enzymes for generating bioactive molecules that do not naturally occur, expanding the scope of biocatalytic strategies for drug discovery and natural product modification.