Indigo Formation as a Predictor of Substrate Promiscuity in Cytochrome P450 BM3 Libraries

Cytochrome P450 BM3 natively exhibits substrate promiscuity, mainly in hydroxylation and epoxidation reactions. Whereas directed evolution has broadened its scope of reactivity, screening for specific non-native enzymatic reactivity remains challenging, thereby limiting potential applications. In this study, we investigated the predictive potential of indigo production as an indicator of promiscuous hydroxylation in non-native aromatic compounds. Following site- saturation mutagenesis on 42 residues in the active-site region, we rapidly identified 97 previously unreported variants at 18 unique positions, significantly increasing the database of indigo-positive variants. From this expanded pool, we isolated 80 well-expressed indigo-positive variants and evaluated them alongside 46 well-expressed indigo-negative variants for their hydroxylation activity on the representative non-native aromatic compounds, anisole and naphthalene. We observed a strong correlation between indigo formation and aromatic compound hydroxylation, with 73-80% of indigo-positive variants hydroxylating these compounds, compared to 40-45% of indigo-negative variants. Notably, only indigo-positive variants showed greater than 3-fold higher activity than the wild-type P450 BM3. Furthermore, a positive correlation was obtained between promiscuous hydroxylation of anisole and naphthalene. The approach also allowed identification of new variants that hydroxylate a further aromatic compound, 4-phenyl-2-butanone, suggesting generality. Although we did not succeed in identifying the molecular basis underlying the indigo- positive phenotype, our findings demonstrate that indigo production is an effective screening tool, expediting the discovery of novel functional genes for promiscuous aromatic hydroxylation reactions. This method offers a cost-effective and efficient approach to identify and develop new biocatalysts, advancing the potential applications of P450 BM3 in scientific and industrial fields.