Directed evolution of non-natural cytochrome P450 enzymes:Developing potent biocatalysts and tracing the determinants of enzyme functionality

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Enzymes catalyze chemical reactions with exquisite selectivity and efficiency under mild conditions, making them superior tools for biotechnological applications and eco-friendly green chemistry. However, many industrially important chemical reactions are not catalyzed by any known enzyme, severely limiting the repertoire of chemical compounds accessible to biocatalytic production processes. To overcome this lack of useful enzymes, directed protein evolution has been applied to derive novel enzymes with non-natural catalytic activity. Recently, several novel cytochrome P450 enzymes catalyzing non-natural reactions were obtained with this approach, impressively demonstrating the versatility and evolvability of cytochrome P450 enzymes. However, the novel P450 enzymes frequently show low catalytic activity and low total turnover numbers due to fast enzyme inactivation. This low enzymatic activity prohibits their use as industrial biocatalysts and raises the fundamental question how novel, efficient enzymes develop in nature. I therefore propose to employ directed protein evolution to engineer non-natural cytochrome P450 enzymes catalyzing aziridination reactions towards high catalytic activity in live host cells. Libraries of P450 enzyme mutants will be generated by random mutagenesis and site-directed mutagenesis of selected enzyme residues. The resulting mutant libraries will be screened for P450 variants showing improved catalytic activity in whole live cells. Iterative rounds of mutagenesis and screening will be performed to evolve cytochrome P450 variants catalyzing aziridination reactions at high efficiency and stability in vivo. In a second step, these improved variants will be subjected to detailed biochemical and structural analyses to define the molecular determinants of in vivo enzymatic activity. The proposed research would thereby serve two objectives: First, novel cytochrome P450 enzymes with potent in vivo activity would be obtained, enabling their subsequent employment in biocatalytic production processes. Secondly, insights into the molecular determinants and structure-function relationships of enzyme catalytic activity in vivo will be generated, tracing the evolutionary paths leading from an inefficient enzyme to a highly active variant with potential relevance for engineering of other protein and enzyme families. Thus, the proposed research is expected to advance the field of biocatalysis and contribute to sustainable chemical production processes.