Cytochrome P450 (CYP) monooxygenases, the nature’s most versatile biological catalysts have unique ability to catalyse regio-, chemo-, and stereospecific oxidation of a wide range of substrates under mild reaction conditions, thereby addressing a significant challenge in chemocatalysis. Though CYP enzymes are ubiquitous in all biological kingdoms, the divergence of CYPs in fungal kingdom is manifold. The CYP enzymes play pivotal roles in various fungal metabolisms starting from housekeeping biochemical reactions, detoxification of chemicals, and adaptation to hostile surroundings. Considering the versatile catalytic potentials, fungal CYPs has gained wide range of attraction among researchers and various remarkable strategies have been accomplished to enhance their biocatalytic properties. Numerous fungal CYPs with multispecialty features have been identified and the number of characterized fungal CYPs is constantly increasing. Literature reveals ample reviews on mammalian, plant and bacterial CYPs, however, modest reports on fungal CYPs urges a comprehensive review highlighting their novel catalytic potentials and functional significances. In this review, we focus on the diversification and functional diversity of fungal CYPs and recapitulate their unique and versatile biocatalytic properties. As such, this review emphasizes the crucial issues of fungal CYP systems, and the factors influencing efficient biocatalysis.

Omega hydroxy fatty acids (ω-OHFAs) are multifunctional compounds that act as the basis for the production of various industrial products with broad commercial and pharmaceutical implications. However, the terminal oxygenation of saturated or unsaturated fatty acids for the synthesis of ω-OHFAs is intricate to accomplish through chemocatalysis, due to the selectivity and controlled reactivity in C-H oxygenation reactions. Cytochrome P450, the ubiquitous enzyme is capable of catalyzing the selective terminal omega hydroxylation naturally in biological kingdom. To gain a deep insight on the biochemical role of fungal P450s towards the production of omega hydroxy fatty acids, two cytochrome P450 monooxygenases from Fusarium oxysporum (FoCYP), FoCYP539A7 and FoCYP655C2; were identified, cloned, and heterologously expressed in Saccharomyces cerevisiae. For the efficient production of ω-OHFAs, the S. cerevisiae was engineered to disrupt the acyl-CoA oxidase enzyme and the β-oxidation pathway inactivated (ΔPox1) S. cerevisiae mutant was generated. To elucidate the significance of the interaction of redox mechanism, FoCYPs were reconstituted with the heterologous and homologous reductase systems - S. cerevisiae CPR (ScCPR) and F. oxysporum CPR (FoCPR). To further improve the yield, the effect of pH was analyzed and the homologous FoCYP-FoCPR system efficiently hydroxylated caprylic acid, capric acid and lauric acid into their respective ω-hydroxy fatty acids with 56%, 79% and 67% conversion. Furthermore, based on computational simulations, we identified the key residues (Asn106 of FoCYP539A7 and Arg235 of FoCYP655C2) responsible for the recognition of fatty acids and demonstrated the structural insights of the active site of FoCYPs. Fungal CYP monooxygenases, FoCYP539A7 and FoCYP655C2 with its homologous redox partner, FoCPR constitutes a promising catalyst due to its high regio- and stereo-selectivity in the hydroxylation of fatty acids and in the substantial production of industrially valuable ω-hydroxy fatty acids.

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.

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H₂, and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.

The human cytochrome P450 (CYP) enzyme CYP4A22 is an orphan CYP with unknown function. Here, through functional expression in fission yeast, we show that CYP4A22 catalyzes fatty acid hydroxylation as well as aliphatic or aromatic hydroxylations of luciferin‐based probe substrates. Mechanistic molecular modeling of CYP4A22 suggests that its ω‐hydroxylation activity is hampered by a more spacious active site compared to CYP4B1. Substrate recognition via side‐chains R96 and R233 is indicated by dynamic three‐dimensional pharmacophores (dynophores) derived from molecular dynamics simulations. CYP4A22 activity is inhibited by three unspecific CYP inhibitors. A comparison of CYP4A22*1 (the reference standard sequence) with CYP4A22‐WT (the most common allele) revealed that for the four substrates tested the WT‐enzyme always had lower activity.

The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole.