Cytochromes P450 as useful biocatalysts: addressing the limitations

Abstract

Cytochrome P450 monooxygenases (P450s or CYPs) are a unique family of enzymes which are capable of catalysing the regio- and stereospecific oxidation of non-functionalised hydrocarbons. Despite the enormous synthetic potential of P450s, these enzymes have yet to be extensively employed for research purposes or in industry. Lack of stability, low activity, narrow substrate specificity, expensive cofactor requirements, limited solvent tolerance and electron supply are some of the main reasons why the academic and industrial implementation of these important biocatalysts remains a challenge. Considering the significance of P450s, many research groups have focused on improving their properties in an effort to make more robust catalysts with broad synthetic applications. This article focuses on some of the factors that have limited the exploitation of P450s and explores some of the significant steps that have been taken towards addressing these limitations.

Combinatorial Engineering of Yarrowia lipolytica as a Promising Cell Biorefinery Platform for the de novo Production of Multi-Purpose Long Chain Dicarboxylic Acids

“…Regeneration of NADPH is crucial in whole-cell P450-based biotransformation [116], particularly in the NADPH-and adenosine triphosphate (ATP)-consuming process of fatty acid biosynthesis and oxidation. Moreover, whole cells also provide a protected environment for the increased stability of the P450 enzymes [51]. A whole cell biocatalyst is a cheaper option than the isolated or immobilized enzyme [106].…”

Strategies for the construction of insect P450 fusion enzymes

“…P450s do not tolerate solvents well, so many efforts to solubilize the enzymes render them inactive [31]. Because P450s have diverse substrates, the activity of a recombinant enzyme can only be established by screening a library of substrates, some of which are also only sparing soluble [31]. Finally, the industrial application of P450s is also hampered by the need for the expensive electron donor nicotinamide adenine dinucleotide phosphate (NADPH) [31,36,37].…”

“…These could be used to investigate the origin of pesticide resistance and to facilitate the development of new insecticides that inhibit P450s [24]. Furthermore, insect P450s could also be deployed as industrial enzymes for the production of fine chemicals and pharmaceuticals that cannot be produced economically by total chemical synthesis [31] or for the development of new processes based on bioelectrocatalysis [32]. Finally, they could be used for remediation purposes, such as the removal of pesticide residues and other xenobiotics from wastewater and the environment [33].…”

“…Because P450s have diverse substrates, the activity of a recombinant enzyme can only be established by screening a library of substrates, some of which are also only sparing soluble [31]. Finally, the industrial application of P450s is also hampered by the need for the expensive electron donor nicotinamide adenine dinucleotide phosphate (NADPH) [31,36,37]. Catalytically active insect P450s have been expressed in E. coli [38] and insect cell lines [30] but in all cases the yields were insufficient for industrial application.…”

“…The standard approach to determine the catalytic activity of P450s is heterologous expression and reconstitution [22], but the enzymes must be reconstituted in a system containing detergents and phospholipids for solubilization, and the correct CPR components and cofactors must be supplied [34,35]. P450s do not tolerate solvents well, so many efforts to solubilize the enzymes render them inactive [31]. Because P450s have diverse substrates, the activity of a recombinant enzyme can only be established by screening a library of substrates, some of which are also only sparing soluble [31].…”