Rapid identification of cytochrome P450cam variants by in vivo screening of active site libraries

Abstract The selection of cytochrome P450 enzymes from large variant libraries, and the subsequent use of these enzymes in preparative scale biotransformations, remains a formidable challenge due to the complexities of the associated electron transport systems. Here, a powerful approach for the generation and screening of P450 cam libraries for new function is presented that is both flexible and robust. A targeted library was generated wherein only the P450 cam active-site amino acids Y96 and F98 were fully randomized and biotransformations, using a novel P450 cam whole-cell system, were screened by GC–MS for the hydroxylation of diphenylmethane. One in 50 of the reactions screened, including 16 different variants, produced 4-hydroxydiphenylmethane with up to 92% conversion observed in the case of the Y96A variant. These results demonstrate a primary example of the screening of P450 cam libraries in a format that is compatible with extension to preparative scale reactions.