Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi

Published ArticleGenome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes

Genome-wide identification, annotation and characterization of novel thermostable cytochrome P450 monooxygenases from the thermophilic biomass-degrading fungi Thielavia terrestris and Myceliophthora thermophila

Published ArticleCytochrome P450 monooxygenases (P450s) are ubiquitous heme-thiolate proteins that have potential biotechnological application. Thermostable-P450s that can withstand hostile industrial conditions, such as high temperatures, extremes of pH and organic solvents, are needed for biotechnological usage. Here, for the first time, we report a large number of thermostable-P450s from two thermophilic biomass-degrading fungi, Myceliophthora thermophila and Thielavia terrestris. Genome-wide P450 analysis revealed the presence of 79 and 70 P450s (P450ome) in T. terrestris and M. thermophila. Authentic P450s containing both the P450 signature domains (EXXRand CXG) were classified as follows: T. terrestris (50 families and 56 subfamilies) and M. thermophila (49 families and 53 subfamilies). Bioinformatics analysis of P450omes suggested the presence of a large number of thermostable-P450s. Based on aliphatic index cut-off ([90), 14 and 11 P450s were determined to be thermostable in T. terrestris and M. thermophila. Among the thermostable P450s, six P450s from T. terrestris and three from M. thermophila had a melting temperature (Tm) of [65 C, suggesting their hyperthermal tolerance. Analysis of the instability index of two ascomycete P450omes revealed the presence of 12 and 19 in vitro stable P450s in T. terrestris and M. thermophila. Overall, six P450s from T. terrestris and four from M. thermophila showed both thermal tolerance and in vitro stability. Thermophilic ascomycetes P450s are of potential interest from a structural, mechanistic and biotechnological point of view, as five P450s showed higher thermal tolerance and five showed higher in vitro stability compared to the wellcharacterized thermostable-P450s CYP175A1 (bacteria) and CYP119 (archaea)

Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi

<div><p>Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, <i>Phanerochaete chrysosporium</i>, <i>Phanerochaete carnosa</i>, <i>Agaricus bisporus</i>, <i>Postia placenta</i>, <i>Ganoderma</i> sp. and <i>Serpula lacrymans</i>, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of <i>Phanerochaete chrysosporium</i> and <i>Phanerochaete carnosa,</i> suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes.</p></div

Phylogenetic and gene-structure analysis of CYP5146 family.

<p>Eighteen P450 sequences from two white rot basidiomycetes, <i>Phanerochaete chrysosporium</i> (Pchr) and <i>Phanerochaete carnosa</i> (Pcar), were included in the tree. The minimum evolution tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For ease of visual identity, the tree branch color, protein name, protein ID (parenthesis) and model basidiomycete species name were presented in red (Pchr) and pink (Pcar). The protein size in amino acids is shown in the figure. The gene-structure analysis for each P450 was presented in the form of exon-intron organization. A graphical format showing parallel (gene size) and vertical lines (introns) is presented for P450s showing similar gene structure (highlighted with unique color). For the rest of the P450s, the number of exons and introns was shown. The genetic location of P450 is shown in the form of the scaffold number.</p

Minimum evolution tree of CYP512 family.

<p>In total 82 CYP512 P450 sequences were included in the tree. The tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For ease of visual identity, the tree branch color, protein name, protein ID (parenthesis) and species name were presented with unique color. Abbreviations: Pchr, <i>Phanerochaete chrysosporium</i>; Pcar, <i>Phanerochaete carnosa</i>; Abis, <i>Agaricus bisporus</i>; Gsp, <i>Ganoderma</i> sp.; Ppla, <i>Postia placenta</i>; Slac, <i>Serpula lacrymans</i>.</p

Phylogenetic and gene-structure analysis of CYP63 family.

<p>In total 38 CYP63 P450 sequences from six selected model basidiomycetes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086683#pone-0086683-g001" target="_blank">Fig. 1</a>) were included in the tree. The minimum evolution tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For easy of visual identity, the tree branch color, protein name, protein ID (parenthesis) and model basidiomycete species name were presented with unique color. The protein size in amino acids is also shown in the figure. Gene-structure analysis for each P450 was presented in the form of exon-intron organization. A graphical format showing parallel (gene size) and vertical lines (introns) is presented for P450s showing similar gene structure (highlighted with unique background color). For the rest of the P450s the number of exons and introns was shown. The genetic location of P450 is shown in the form of the scaffold number. Abbreviations: Pchr, <i>Phanerochaete chrysosporium</i>; Pcar, <i>Phanerochaete carnosa</i>; Abis, <i>Agaricus bisporus</i>; Gsp, <i>Ganoderma</i> sp.; Ppla, <i>Postia placenta</i>; Slac, <i>Serpula lacrymans</i>.</p

Phylogenetic and gene-structure analysis of CYP5348 family.

<p>In total 47 CYP5348 P450 sequences from two model basidiomycetes, <i>Postia placenta</i> (Ppla) and <i>Ganoderma</i> sp. (Gsp), were included in the tree. The minimum evolution tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For ease of visual identity, the tree branch color, protein name, protein ID (parenthesis) and model basidiomycete species name were presented in blue (Ppla) and magenta (Gsp). The protein size in amino acids is shown in the figure. The gene-structure analysis for each P450 was presented in the form of exon-intron organization. A graphical format showing parallel (gene size) and vertical lines (introns) is presented for P450s showing similar gene structure. For the rest of the P450s, the number of exons and introns was shown. The genetic location of P450 is shown in the form of the scaffold number.</p

Analysis of P450 gene duplications and gene-structure (introns) in P450 families enriched in the basidiomycete species, <i>Phanerochaete chrysosporium</i>, <i>Phanerochaete carnosa</i>, <i>Agaricus bisporus</i>, <i>Ganoderma</i> sp., <i>Postia placenta</i>, and <i>Serpula lacrymans</i>.

a<p>Scaffolds and P450s present on the scaffold were separated by commas. For more information on tandem gene arrangements in scaffold, see figures.</p>b<p>For each P450 family the minimum and maximum number (min-max) of introns observed in member P450s (≥400 amino acid length) were presented. A number of P450s showing the same number of introns in their gene-structure (P/I) are also presented in the table.</p><p>Abbreviations: FA, Family is absent in the fungus; NA, not applicable owing to the presence of single or double copies of P450 genes and P450s with short amino acid length.</p

Phylogenetic and gene-structure analysis of CYP5141 family.

<p>Thirty-two CYP5141 P450 sequences from six model basidiomycetes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086683#pone-0086683-g001" target="_blank">Fig. 1</a>) were included in the tree. The minimum evolution tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For ease of visual identity, the tree branch color, protein name, protein ID (parenthesis) and model basidiomycete species name were presented with unique color. The protein size in amino acids is also shown in the figure. The gene-structure analysis for each P450 was presented in the form of exon-intron organization. A graphical format showing parallel (gene size) and vertical lines (introns) is presented for P450s showing similar gene structure. For the rest of the P450s, the number of exons and introns was shown. The genetic location of P450 is shown in the form of the scaffold number. Abbreviations: Pchr, <i>Phanerochaete chrysosporium</i>; Pcar, <i>Phanerochaete carnosa</i>; Abis, <i>Agaricus bisporus</i>; Gsp, <i>Ganoderma</i> sp.; Ppla, <i>Postia placenta</i>; Slac, <i>Serpula lacrymans</i>.</p

Phylogenetic analysis of CYP5144 family.

<p>In total 128 CYP5144 P450 sequences from six model basidiomycetes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086683#pone-0086683-g001" target="_blank">Fig. 1</a>) were included in the tree. The minimum evolution tree was constructed using the close-neighbor-interchange algorithm in MEGA (version 5.05). For ease of visual identity, the tree branch color, protein name, protein ID (parenthesis) and model basidiomycete species name were presented with unique color. Abbreviations: Pchr, <i>Phanerochaete chrysosporium</i>; Pcar, <i>Phanerochaete carnosa</i>; Abis, <i>Agaricus bisporus</i>; Gsp, <i>Ganoderma</i> sp.; Ppla, <i>Postia placenta</i>; Slac, <i>Serpula lacrymans</i>.</p