The model white rot fungus Phanerochaete chrysosporium has the extraordinary ability to degrade (to CO2) lignin and detoxify a variety of chemical pollutants. Whole genome sequencing of this fungus has revealed the presence of the largest P450ome in fungi comprising approx. 150 P450 genes, most of which have unknown function. On the basis of our genome-wide structural and evolutionary analysis, these P450 genes could be classified into 12 families and 23 subfamilies and under 11 fungal P450 clans. The analysis further revealed an extensive gene clustering with a total of 16 P450 clusters constituted of up to 11 members per cluster. In particular, evidence and role of gene duplications and horizontal gene transfer in the evolution of these P450 clusters have been discussed using two of the P450 families [CYP63 and CYP505 (where CYP is cytochrome P450)] as examples. In addition, the observed differential transcriptional induction of the clustered members of the CYP63 gene family, in response to different xenobiotic chemicals and carbon sources, indicated functional divergence within the P450 clusters, of this basidiomycete fungus.
The basidiomycetous white rot fungus Phanerochaete chrysosporium, originally known to degrade lignin and other plant cell-wall components as a part of the Nature's carbon cycle, has been shown to possess an extraordinary ability to oxidize and detoxify a broad range of toxic chemical pollutants. Initially, ligninolytic peroxidases were thought to be primarily involved in the lignin and xenobiotic oxidation processes under secondary metabolic (nutrient-limited) conditions. However, subsequent lines of evidence based on oxidizability of xenobiotic chemicals in the absence of peroxidase expression [1–4] as well as recent sequencing of the P. chrysosporium genome have led to the increased interest in alternate oxygenases particularly P450 mono-oxygenases, in this versatile xenobiotic biodegrader and detoxifier.
Cytochrome P450 mono-oxygenases (‘P450s’) are a superfamily of haem-thiolate proteins that are involved in the metabolism of a wide variety of endogenous and xenobiotic compounds. P450 enzymes are found in different life forms including prokaryotes (archaea and bacteria), lower eukaryotes (fungi and insects) and higher eukaryotes (plants and animals including humans). A typical eukaryotic P450 system primarily consists of a P450 mono-oxygenase and a POR (NADPH:P450 oxidoreductase), both associated with membranes in the endoplasmic reticulum. However, soluble bacterial P450-like P450 enzymes such as P450foxy have been characterized in fungi . Although the levels of protein identity between the different P450 mono-oxygenases may be as low as 16%, the primary structural folds have been maintained over the course of evolution. P450 proteins are classified primarily based on amino acid similarity, with less than 40% similarity defining a family and 40–55% defining a subfamily. However, classification based on clan, a higher order of grouping, is lately gaining increased acceptance in the P450 community [6,7].
Based on the recent whole genome sequencing by the JGI (Joint Genome Institute) of the U.S. Department of Energy, P. chrysosporium has been shown to possess the highest number of P450 sequences among fungi (http://drnelson.utmem.edu/cytochromeP450.html). Only plants seem to possess a higher number of P450s than the white rot fungi . Our genome-wide analysis of the P. chrysosporium P450ome lately suggested the presence of extensive P450 gene clustering in this genome . Furthermore, our global P450 gene transcriptional profiling under two distinct nutrient conditions, nutrient-limited (ligninolytic) and nutrient-sufficient (non-ligninolytic), revealed a functional divergence in this P450ome . In the present paper, we present a summarized account of our in depth structural and evolutionary analysis of the P450ome in terms of the P450 gene clusters with a focus on CYP63 (where CYP is cytochrome P450) and CYP505, and demonstrate extensive functional divergence within the CYP63 P450 clusters as an example based on differential gene expression/induction in response to a wide variety of nutrients and xenobiotics.
P450ome structure in P. chrysosporium
P450 mono-oxygenases and their classification in P. chrysosporium (P450ome)
The P450 genes (P450ome) in this organism constitute nearly 1% of its genome coding sequence , most of which have unknown function. Of the 163 originally predicted P450 sequences, 108 have been assembled full-length and tentatively annotated based on general overall sequence homology analysis (http://drnelson.utmem.edu/cytochromeP450.html, ). Based on phylogenetic grouping of the predicted 163 P450 sequences, 26 phylogenetic clusters were initially reported by our laboratory . Our further phylogenetic analysis of the 126 full-length or near full-length (>300 amino acids) P450 genes coupled with standard sequence homology criterion had shown that the white rot P450s fall into 12 families and 23 subfamilies . A comprehensive diagram showing the relative number of genes in each of the 12 families is shown in Figure 1. The gene number per family varied from as low as one (CYP51, CYP61 and CYP62) to as high as 54 (CYP64). Genome-wide structural analysis revealed an extensive P450 gene clustering with as many as 16 P450 gene clusters. The highest number of clusters belonged to the CYP64 family (seven clusters located on scaffold 24), followed by CYP503 (three clusters located on scaffold 30), CYP67 (two clusters located one each on scaffold 53 and scaffold 97), CYP505 (two clusters located on scaffold 73), CYP58 (one cluster on scaffold 79) and CYP63 (one cluster on scaffold 20) respectively.
P450ome of the white rot fungus P. chrysosporium
Structural analysis of CYP63 gene family
CYP63, the first P450 family discovered in white rot fungi in the pre-genomic efforts, has been the focus of research in our laboratory. This family consists of seven P450 genes (pc-1–pc-7). We reported isolation of the first two full-length tandemly linked P450 genes pc-1 and pc-2 of the CYP63 family and cloning of cDNA and a splice variant cDNA of pc-1 . Further sequencing of the genomic clones containing the two tandem genes (pc-1 and pc-2) led to the isolation of an additional linked gene pc-3 . Subsequently, full-length cDNAs for pc-2 and pc-3 were cloned . PC-3 protein showed high amino acid homology with PC-2 (85.2%) and PC-1 (58.9%). The three CYP63 genes, pc-1 (CYP63A1), pc-2 (CYP63A2) and pc-3 (CYP63A3) are tandemly linked genes with shorter intergenic regions (322 and 469 bp), and form a tight cluster of genes on scaffold 20. The other four genes of this family (pc-4–pc-7) are scattered on scaffolds 151 (pc-4), 101 (pc-5 and pc-6) and 57 (pc-7) in the genome (Figure 2). The subfamily assignment is as follows: pc-4 under CYP63A, pc-5 and pc-6 under CYP63B and pc-7 under the subfamily CYP63C.
CYP63 gene family: structural features and gene clusters of the member P450 genes
Our structural analysis revealed that members of the same P450 cluster (genes found on the same scaffold) mostly have a similar gene structure , whereas the member genes of the same CYP family may vary in their gene structure (intron/exon ratio), albeit to a varying extent. For instance, the CYP63 family showed the presence of two gene clusters, one consisting of pc-1, pc-2 and pc-3, and the other consisting of pc-5 and pc-6. Intron–exon organization within each cluster was similar. On the other hand, the CYP63 family as a whole showed a wider range in terms of intron–exon structure, with the lowest number of introns in pc-7 (six introns) and the highest number in pc-1 (14 introns), as diagrammatically shown in Figure 2. Likewise, the protein sizes of the members from the CYP63 family varied between 571 amino acids (PC-7) and 603 amino acids (PC-3). Nevertheless, all these proteins had the relatively conserved P450 motifs, haem-binding region (HR2), helix-I, and helix-K, and the N-terminal transmembrane domain (centred around 21–57 amino acids), the typical characteristics of the eukaryotic membrane P450s.
Evolution of the P450ome in P. chrysosporium
Conventionally, family level classification has been primarily used for grouping the different P450 enzymes. However, this method of classification does not reveal the evolutionary origin of these genes. For instance, similar P450 genes are classified into different families in different organisms that could have otherwise originated from a common ancestor . In order to address this issue, a higher order of grouping (Clan level) was introduced . A clan represents a group of P450 families across species. So far, there are nine clans in vertebrates and ten in plants. Clan level comparisons revealed that 12 P450 families of P. chrysosporium have resemblances to 11 known fungal P450 clans. Furthermore, they showed varying degrees of similarity to the P450 genes from different ascomycetous fungi such as Aspergillus and Fusarium, thus suggesting that P450 genes in this basidiomycetous fungus are acquired as a part of the vertical descent from a common ancestor followed by further diversification . For instance, the CYP63 family of proteins (pc-1–pc-7) have structural resemblance to the CYP52 family of ascomycetous yeasts and have therefore been classified under the CYP52 clan . This structural relationship between the CYP52 family of yeasts and the CYP63 family of proteins in P. chrysosporium appears conserved to a certain extent even at the functional level based on our observations that, like CYP52, the CYP63 genes pc-1, pc-2 and pc-3 are also inducible by alkanes [13,14,16]. However, further structural diversification of the CYP63 genes seems to have occurred during the evolutionary process, giving them additional functional roles, as compared with CYP52.
Evidence for gene duplications and translocations
The clustered CYP63 family members pc-1, pc-2 and pc-3 tandemly linked on a chromosome have a conserved structural organization in terms of the location and number of introns. The overall amino acid homology among the three members is high, with PC-2 and PC-3 being closer (85.2%) with each other than with PC-1 (58.9%). Alternative splicing variants observed in pc-1  raise the possibility of occurrence of similar variants in the other two members (pc-2 and pc-3) of this cluster in view of their common intron–exon organization. This likelihood was further indicated by the observation in our microarray experiments that multiple probes designed over the entire length of the gene gave different levels of hybridization (results not shown) than one single probe. Further, the three members of this cluster (pc-1, pc-2 and pc-3) are closer to each other in terms of percentage identity at the protein level, as compared with the other four members in this family. These multiple lines of evidence suggest that this CYP63 cluster arose by a simple gene duplication event. In another cluster of the CYP63 family, comprising the non-tandem genes pc-5 and pc-6, a similar level of deduced amino acid homology (76.9%) was observed. The two member genes (pc-5 and pc-6) have high gene-structure conservation and homologous flanking sequences, again suggesting that the cluster formation may have involved gene duplication and translocation events mediated by the flanking sequences. On the other hand, pc-7 which has overall similarity with pc-5 and pc-6 based on the position of introns, differs significantly at the protein level possibly because of loss of three of the introns (introns 1, 5 and 8) and consequent frame-shift(s), after the duplication event. This suggests the occurrence of an additional step, a deletion, during evolution following gene duplication. In this context, pc-4 seems to have evolved as a result of the loss of four of the conserved introns (intron 3, 4, 10 and 13) and acquisition or shift in the two introns (introns 7 and 14), when compared with pc-1.
Evidence of HGT (horizontal gene transfer)
In the P. chrysosporium P450ome, another interesting set of enzymes is the P450foxy-like proteins. Primarily identified in the bacterium Bacillus megaterium  and later isolated from the ascomycete fungus Fusarium oxysporum , these proteins are a fusion between a P450 mono-oxygenase gene and a P450 reductase gene. The P450foxy-like proteins from the basidiomycete P. chrysosporium can be classified into the CYP505 clan based on their overall similarity to the known ascomycete P450foxy proteins . However, no other basidiomycete fungus sequenced so far has been found to have the P450foxy-like proteins. The only fungal class previously known to possess the P450foxy proteins is the ascomycetes. This raises an interesting question as to how these two different classes of fungi (ascomycetes and basidiomycetes) have acquired similar genes. This phenomenon can be explained based on the concept of HGT, which is the process of exchange of genetic material between distantly related species. Although this phenomenon seems to be more commonly known in prokaryotes, probably due to the more extensive experimental and genome sequence data available in prokaryotes as compared with eukaryotes [18,19], there is evidence to show the occurrence of HGT in plants and in some phagotrophic eukaryotes [20,21]. However, the understanding on this phenomenon is changing with the increase in the burgeoning eukaryotic genome data becoming available in recent years. Based on our systematic genomic analysis of P450foxy-like proteins in P. chrysosporium , it can be hypothesized that one such horizontal transfer event has occurred from an ascomycete to the basidiomycete fungus immediately after the ascomycete–basidiomycete split approx. 400 million years ago.
Xenobiotic induction of individual members of the CYP63 family
Functional diversity of the P450ome gene clusters as evidenced by differential regulation of expression
Differential induction of P450 genes in clusters by xenobiotics
In order to understand the differential induction of P450 genes in genomic clusters, CYP63 family was studied as an example. For this, P. chrysosporium cultures were treated with a range of xenobiotics under nutrient-sufficient culture conditions using ME (malt extract) broth. Transcriptional induction pattern for four of the CYP63 genes (pc-4–pc-7) was compared with the induction pattern of the three tandemly linked genes pc-1, pc-2 and pc-3 (reported earlier) based on real-time quantitative RT (reverse transcriptase)–PCR analysis using gene-specific primers. The results for the induction studies have been summarized in Figure 3. All seven genes showed transcriptional induction with alkanes and its derivatives, albeit to a varying extent. This conforms with the structurally predicted substrates (alkanes) for the CYP63 family of proteins . On exposure to the other xenobiotics, pc-1 showed transcriptional induction in response to mono-aromatics (m-hydroxy benzoic acid, nitrophenol and phenoxy acetic acid), polycyclic aromatic compounds (naphthalene, phenanthrene, pyrene, 3-methylcholanthrene, p-p′-biphenol and polychlorinated biphenyl), alkyl-substituted aromatics (dodecylbenzene sulfonate, 1-phenyl dodecane and limonene) and certain classical P450 inducers such as oestradiol. pc-2 on the other hand showed no induction in response to mono-aromatic compounds, although polycyclic aromatic compounds did induce the expression, and so did the alkyl-substituted aromatics. pc-3 showed low levels of induction in response to both the mono-aromatics and polycyclic aromatics but none or negligible induction in response to alkyl-substituted aromatics and the classical P450 inducers. pc-4 showed induction specifically in response to polycyclic aromatic compounds and alkyl-substituted aromatics. pc-5 showed no induction in response to either mono-aromatic or polycyclic aromatic compounds and only negligible induction in response to alkyl-substituted aromatics. pc-6 showed negligible levels of induction in response to mono-aromatics, high levels of induction in response to some of the tested polycyclic aromatic compounds (1-naphthol and 1,2-benzanthracene) and several alkyl-substituted aromatic compounds. pc-7 showed very poor induction in response to both mono and polycyclic aromatic compounds and high levels of induction in response to some of the tested alkyl-substituted aromatic compounds (alkyl phenols).
Nutrient regulation of expression of genes in P450 clusters
We further studied the regulation of expression of the clustered CYP63 genes in response to several carbohydrate sources using LN (low nitrogen) culture conditions. As reported earlier, pc-1 expression was comparable for glucose, sucrose and raffinose, whereas it was relatively lower for the complex polysaccharides, starch and cellulose. pc-2 expression on the other hand was not significantly affected by the different carbon sources . pc-3 showed highest expression in the presence of starch, whereas cellulose supported the highest expression of pc-4 and pc-5. pc-6 showed highest expression in sucrose and xylan, whereas pc-7 showed comparable expression in all six test carbon sources. The pattern of expression of these genes was completely different when HN (high nitrogen) culture conditions were tested. This suggested that, although belonging to the same family of proteins and some of them having very high levels of sequence similarity among them (especially pc-1, pc-2 and pc-3), the genes in clusters are independently regulated and probably have diverse catalytic functions, with a substrate range wider than that predicted based on structural homologies.
In conclusion, the extraordinary P450ome of the white rot fungus is characterized by the presence of extensive gene clustering, with 16 clusters of varying composition (2–11 member genes). While most of these gene clusters seem to have evolved vertically from ancestor fungi, there is evidence of HGT from ascomycetes for one of the P450 families (P450foxy family). The gene diversity of the P450ome, particularly within the P450 gene clusters, appears to have evolved as a result of gene duplication and translocation events. Functional divergence within the P450 gene clusters was demonstrated in our studies based on differential regulation of expression/induction even among member genes of structurally homogenous P450 clusters such as of the CYP63 family. This level of structural and functional diversity in the P450ome that has arisen by extensive gene evolution could be the underlying factor for the versatility of this white rot fungus in the detoxification and biodegradation of a broad range of xenobiotic chemicals.
8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology: Independent Meeting held at Swansea Medical School, Swansea, Wales, U.K., 23–27 July 2006. Organized and Edited by D. Kelly, D. Lamb and S. Kelly (Swansea, U.K.).