Functional expression and regulation of eukaryotic cytochrome P450 enzymes in surrogate microbial cell factories

Abstract

Cytochrome P450 (CYP) enzymes play crucial roles during the evolution and diversification of ancestral monocellular eukaryotes into multicellular eukaryotic organisms due to their essential functionalities including catalysis of housekeeping biochemical reactions, synthesis of diverse metabolites, detoxification of xenobiotics, and contribution to environmental adaptation. Eukaryotic CYPs with versatile functionalities are undeniably regarded as promising biocatalysts with great potential for biotechnological, pharmaceutical and chemical industry applications. Nevertheless, the modes of action and the challenges associated with these membrane-bound proteins have hampered the effective utilization of eukaryotic CYPs in a broader range. This review is focused on comprehensive and consolidated approaches to address the core challenges in heterologous expression of membrane-bound eukaryotic CYPs in different surrogate microbial cell factories, aiming to provide key insights for better studies and applications of diverse eukaryotic CYPs in the future. We also highlight the functional significance of the previously underrated cytochrome P450 reductases (CPRs) and provide a rational justification on the progression of CPR from auxiliary redox partner to function modulator in CYP catalysis.

Keywords: Cytochrome P450 enzymes; Cytochrome P450 reductase; Electron transfer; Heterologous expression; Membrane-bound proteins; Microbial cell factories; N-terminal transmembrane domain; Redox partners.

Fig. 1

Eukaryotic CYP catalytic cycle and the electron transfer mechanism of CPR/Cyt B5/CB5R redox system.

Fig. 2

Diversity of eukaryotic CYP systems based on the topology of protein components. A. Class I (mitochondrial: NADPH→[AdR]→[Adx]→[P450]); B. Class II microsomal A: NADPH→[CPR]→[P450]; C. Class II microsomal B: NADPH→[CPR]→[Cyt B5] →[P450]; D. Class II microsomal C: NADH→[CBR]→[Cyt B5]→[P450]; E. Class VIII (NADPH→[CPR-P450]); F. Class IX (NADH→[P450]); and G. Class X [P450] systems.

Fig. 3

Challenges associated with the prokaryotic and eukaryotic expression of membrane-bound eukaryotic CYPs.

Fig. 4

Key approaches to improve eukaryotic CYP production in surrogate cell factory.

Fig. 5

Schematic representation of CYP-CPR reconstitution with different sources of origin. A. CYP reconstitution with endogenous CPR, B. CYP reconstitution with homologous CPR, and C. CYP reconstitution with heterologous CPR.