Engineering cytochrome P450 biocatalysts for biotechnology, medicine and bioremediation

Abstract

Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: i) synthesis of novel drugs and drug metabolites; ii) targeted cancer gene therapy; iii) biosensor design; and iv) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH.

Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of the above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency and utilization of alternate oxidants.

What the reader will gain: The review provides a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine and bioremediation.

Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists and healthcare providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts.

Figure 1

The figure shows 1) application (bottom panel), 2) approach (middle panel), and 3) examples of engineered P450s for desired characteristics (top panel). The application in: 1) biotechnology includes the synthesis of drug metabolites by using a fermentation process and freeze-dry methods; 2) medicine includes activation of the anti-cancer prodrugs by employing GDEPT and monitoring via biosensors drug levels in blood plasma, and 3) bioremediation of environmental pollutants by applying immobilization methods and transgenic plants. The approaches to engineer P450s are: 1) rational/CSM (knowledge-based) and 2) directed evolution (random). The representative examples of engineered P450 enzymes include: 1) CYP2B1 with enhanced activity and stability, 2) CYP3A4 with enhanced activity and utilization of peroxides, and 3) CYP2B6 with enhanced expression and solubility. The figure was synthesized from the following multiple sources, The figures for freeze-dry method, biosensor design, immobilization, and transgenic plants are reproduced from references 30, 51, 31, and 79, respectively, upon request for permission from the respective sources. The Figure for GDEPT was provided by Dr. David Waxman from Boston University. The figures for directed evolution, rational, CSM, 2B1, 3A4, and 2B6 were reproduced from references 83, 140, 89, 94, 95, and 137, respectively.