Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs)

Abstract

Fungi, particularly the white rot basidiomycetes, have an extraordinary capability to degrade and/or mineralize (to CO₂) the recalcitrant fused-ring high molecular weight (⩾4 aromatic-rings) polycyclic aromatic hydrocarbons (HMW PAHs). Despite over 30 years of research demonstrating involvement of P450 monooxygenation reactions in fungal metabolism of HMW PAHs, specific P450 monooxygenases responsible for oxidation of these compounds are not yet known. Here we report the first comprehensive identification and functional characterization of P450 monooxygenases capable of oxidizing different ring-size PAHs in the model white rot fungus Phanerochaete chrysosporium using a successful genome-to-function strategy. In a genome-wide P450 microarray screen, we identified six PAH-responsive P450 genes (Pc-pah1–Pc-pah6) inducible by PAHs of varying ring size, namely naphthalene, phenanthrene, pyrene, and benzo(a)pyrene (BaP). Using a co-expression strategy, cDNAs of the six Pc-Pah P450s were cloned and expressed in Pichia pastoris in conjunction with the homologous P450 oxidoreductase (Pc-POR). Each of the six recombinant P450 monooxygenases showed PAH-oxidizing activity albeit with varying substrate specificity towards PAHs (3–5 rings). All six P450s oxidized pyrene (4-ring) into two monohydroxylated products. Pc-Pah1 and Pc-Pah3 oxidized BaP (5-ring) to 3-hydroxyBaP whereas Pc-Pah4 and Pc-Pah6 oxidized phenanthrene (3-ring) to 3-, 4-, and 9-phenanthrol. These PAH-oxidizing P450s (493–547 aa) are structurally diverse and novel considering their low overall homology (12–23%) to mammalian counterparts. To our knowledge, this is the first report on specific fungal P450 monooxygenases with catalytic activity toward environmentally persistent and highly toxic HMW PAHs.

Introduction

Fused-ring polycyclic aromatic hydrocarbons (PAHs) constitute an important group of highly toxic and significant environmental chemicals that are generated from different anthropogenic and industrial processes and accidents such as crude oil spills [1]. The persistence and genotoxicity of PAHs increase with increasing aromatic rings. Consequently, high molecular weight (HMW) PAHs (⩾4 benzene rings) are particularly a significant problem both from the point of view of environmental clean up as well as human health as these are recalcitrant to biodegradation [2] and are mutagenic and/or carcinogenic to living systems [3]. Among the microorganisms (bacteria, yeasts, and filamentous fungi) investigated for their ability to break down these hazardous chemicals [1], [4], [5], only a few species belonging to actinomycetes and fungi have the ability to oxidize high molecular weight (HMW) PAHs. In particular, white rot group of basidiomycete fungi have shown an extraordinary capability to completely degrade (to CO₂) both low molecular weight (LMW) and HMW PAH compounds [5], [6], [7].

Phanerochaete chrysosporium (henceforth abbreviated as PC) is the most intensively studied model white rot fungus for mechanisms to degrade lignin and a wide range of xenobiotics including PAHs [6]. Originally, its aromatic ring-oxidizing activity was ascribed to the non-specific extracellular peroxidases [8], [9] that are differentially expressed under nutrient-limited (ligninolytic) culture conditions [10]. Subsequent studies by us and others have led to increasing evidences on peroxidase-independent degradation of several aromatics including PAHs under nutrient-sufficient (non-ligninolytic) culture conditions and involvement of P450 monooxygenation reactions [11], [12], [13], [14], [15], [16], [17]. Role of P450 monooxygenation in the rate-limiting initial oxidation of HMW PAHs and certain LMW PAHs with high ionization potential (>7.35 eV) such as phenanthrene has also been reported in other basidiomycete and non-basidiomycete fungi [4], [18]. However, despite the past over 30 years of research on PAH biodegradative fungal species [1], [4], [5], the specific P450 monooxygenase enzymes responsible for this oxidation activity have not been reported. Collectively considering the above discussion, there is a critical need for identification of specific fungal P450 genes and enzymes responsible for the oxidation of HMW and/or both LMW and HMW PAH compounds, in order to understand the P450-mediated mechanisms and to facilitate development of improved biotransformation processes and applications.

Cytochrome P450 monooxygenases are a superfamily of heme-thiolate proteins that catalyze a broad range of reactions such as carbon hydroxylation, heteroatom oxygenation, dealkylation, epoxidation, reduction, dehalogenation [19]. The typical eukaryotic P450 monooxygenase system contains a P450 monooxygenase and a P450 oxidoreductase (POR), both of which are normally membrane-associated. This family of proteins has seen a tremendous growth in the genomic era [[20], http://drnelson.uthsc.edu/CytochromeP450.html]. However, post-genomic functional characterization of orphan P450s in fungal and other genomes has been a challenging task due to their poor amenability to heterologous expression in simpler hosts. Whole genome sequencing of PC [21] has uncovered the presence of a large P450 diversity, comprised of about 150 P450 genes [22]. While functional genomic studies on these P450s are emerging [16], [23], [24], majority remain orphan with virtually unknown function. Hence, there is a great need to characterize the role of individual P450s in this model organism. Our recent studies using the first custom-designed genome-wide P450 microarray [25], [26], have led to a working hypothesis that substrate specific inducibility could be key to identifying the xenobiotic substrates for orphan P450 enzymes in this organism [16], [23], [25]. In this study, we therefore employed a two-stage genome-to-function strategy, including a genome-wide (microarray-based) transcriptional induction profiling to identify candidate P450 genes responsive to PAHs of varying ring-size followed by co-expression and catalytic characterization of the identified recombinantly expressed P450 proteins. These efforts led to identification of a set of six PAH-responsive P450 monooxygenase genes in the PC genome (designated Pc-Pah1 through Pc-Pah6). Subsequent co-expression along with the reductase partner and catalytic analysis revealed their diverse PAH substrate specificity particularly towards HMW (4–5 ring) PAHs. Part of the data reported in this manuscript was presented at the 104th annual general meeting of the American Society for Microbiology, New Orleans, LA [27].