Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes
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Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.
KeywordsAspergillus niger Beauveria bassiana Entomopathogenic fungi Hydrocarbon degradation Metarhizium anisopliae Penicillium chrysogenum
We thank C. Lopez Lastra for kindly providing the M. anisopliae isolate CEP 120 used in this study. We also thank Juan Cruz Ponce for the experimental assistance.
This research was partially supported by CONICET (PIP 112 20110100391) to MCNS and ANPCyT (PICT 2012 1964) to NP. MCNS, JRG, and NP are members of the CONICET Researcher’s Career, Argentina.
- 4.Silva, I. S., Santos, E. C., Menezes, C. R., Faria, A. F., Franciscon, E., Grossman, M., & Durrant, L. R. (2009). Bioremediation of a polyaromatic hydrocarbon contaminated soil by native soil microbiota and bioaugmentation with isolated microbial consortia. Bioresource Technology, 100, 4669–4675.CrossRefGoogle Scholar
- 9.Pedrini, N., Crespo, R., & Juárez, M. P. (2007). Biochemistry of insect epicuticle degradation by entomopathogenic fungi. Comparative Biochemistry and Physiology, 146C, 124–137.Google Scholar
- 10.Pedrini, N., Ortiz-Urquiza, A., Huarte-Bonnet, C., Zhang, S., & Keyhani, N. O. (2013). Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction. Frontiers in Microbiology, 4, 24.CrossRefGoogle Scholar
- 12.Romero, M. C., Urrutia, M. I., Reinoso, H. E., & Moreno-Kiernan, M. (2010). Benzo[a]pyrene degradation by soil filamentous fungi. Journal of Yeast and Fungal Research, 1, 25–29.Google Scholar
- 13.Jerina, D. M., Kaubisch, N., & Daly, J. W. (1971). Arene oxides as intermediates in the metabolism of aromatic substrates: alkyl and oxygen migrations during isomerization of alkylated arene oxides. Proceedings of the National Academy of Sciences of the United States of America, 68, 2545–2548.CrossRefGoogle Scholar
- 15.Zhang, S., Widemann, E., Bernard, G., Lesot, A., Pinot, F., Pedrini, N., & Keyhani, N. O. (2012). CYP52X1, representing new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in entomopathogenic fungus Beauveria bassiana. Journal of Biological Chemistry, 287, 13477–13486.CrossRefGoogle Scholar
- 17.Tanaka, A., & Fukui, S. (1989). Metabolism of n-alkanes. In A. Tanaka & S. Fukui (Eds.), The yeast, 2nd edition (Vol. 3, pp. 261–287). New York: Academic Press.Google Scholar
- 18.Nelson, D. R. (2009). The cytochrome P450 homepage. Human Genomics, 4, 59–65.Google Scholar
- 20.Jawallapersand, P., Mashele, S. S., Kovacic, L., Stojan, J., Komel, R., Pakala, S. B., Krasevec, N., & Syed, K. (2014). Cytochrome P450 monooxygenase CYP53 family in fungi: comparative structural and evolutionary analysis and its role as a common alternative anti-fungal drug target. PloS One, 9(9), e107209.CrossRefGoogle Scholar
- 21.Craft, D. L., Madduri, K. M., Eshoo, M., & Wilson, C. R. (2003). Identification and characterization of the CYP52 family of Candida tropicalis ATCC 20336, important for the conversion of fatty acids and alkanes to α,ω-dicarboxylic acids. Applied and Environmental Microbiology, 69, 5983–5991.CrossRefGoogle Scholar
- 29.McCammick, E. M., Gomase, V. S., Timson, D. J., McGenity, T. J., & Hallsworth, J. E. (2010). Water-hydrophobic compound interactions with the microbial cell. In K. N. Timmis (Ed.), Handbook of hydrocarbon and lipid microbiology–hydrocarbons, oils and lipids: Diversity, properties and formation (Vol. 2, pp. 1451–1466). New York: Springer.CrossRefGoogle Scholar
- 31.Lobo, L. S., Luz, C., Fernandes, E. K. K., Juárez, M. P., & Pedrini, N. (2015). Assessing gene expression during pathogenesis: use of qRT-PCR to follow toxin production in the entomopathogenic fungus Beauveria bassiana during infection and immune response of the insect host Triatoma infestans. Journal of Invertebrate Pathology, 128, 14–21.CrossRefGoogle Scholar
- 32.Roberts, D. W., Gupta, S., & St. Leger, R. J. (1992). Metabolite production by entomopathogenic fungi. Pesquisa Agropecuária Brasileira, 27, 325–347.Google Scholar
- 34.Pedrini, N., Zhang, S., Juárez, M. P., & Keyhani, N. O. (2010). Molecular characterization and expression analysis of a suite of cytochrome P450 enzymes implicated in insect hydrocarbon degradation in the entomopathogenic fungus Beauveria bassiana. Microbiology, 156, 2549–2557.CrossRefGoogle Scholar
- 38.Halo, L. M., Heneghan, M. N., Yakasai, A. A., Song, Z., Williams, K., Bailey, A. M., Cox, R. J., Lazarus, C. M., & Simpson, T. J. (2008). Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana. Journal of the American Chemical Society, 130(52), 17988–17996.CrossRefGoogle Scholar
- 39.Yadav, J. S., Soellner, M. B., Loper, J. C., & Mishra, P. K. (2003). Tandem cytochrome P450 monooxygenase genes and splice variants in the white rot fungus Phanerochaete chrysosporium: cloning, sequence analysis, and regulation of differential expression. Fungal Genetics and Biology, 38, 10–21.CrossRefGoogle Scholar
- 41.Doddapaneni, H., Subramanian, V., & Yadav, J. S. (2005). Physiological regulation, xenobiotic induction, and heterologous expression of P450 monooxygenase gene pc-3 (CYP63A3), a new member of CYP63 gene cluster in the white rot fungus Phanerochaete chrysosporium. Current Microbiology, 50, 292–298.CrossRefGoogle Scholar
- 42.Syed, K., Porollo, A., Lam, Y. W., Grimmett, P. E., & Yadav, J. S. (2013). CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes. Applied and Environmental Microbiology, 79, 2692–2702.CrossRefGoogle Scholar