International Journal of Environmental Bioremediation & Biodegradation
ISSN (Print): 2333-8628 ISSN (Online): 2333-8636 Website: http://www.sciepub.com/journal/ijebb Editor-in-chief: Apply for this position
Open Access
Journal Browser
Go
International Journal of Environmental Bioremediation & Biodegradation. 2016, 4(3), 85-92
DOI: 10.12691/ijebb-4-3-3
Open AccessArticle

Gene Expression Profiling and Enzymatic Function of Phanerochaete Chrysosporium Cytochrome P450s Involved in the Metabolism of Benzo(a)pyrene

Chigu Nomathemba Loice1, , Hirosue Sinji2, Nakamura Chie2, Teramoto Hiroshi2, Ichinose Hirofumi2 and Wariishi Hiroyuki1, 3

1Graduate School of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

2Bioresource chemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan

3Bioresource chemistry, Faculty of Agriculture, Kyushu University, Fukuoka, Japan;Bio-Architecture centre, Kyushu University, Fukuoka, Japan;Innovation Centre for Medical Redox Navigation, Fukuoka, Japan

Pub. Date: December 21, 2016

Cite this paper:
Chigu Nomathemba Loice, Hirosue Sinji, Nakamura Chie, Teramoto Hiroshi, Ichinose Hirofumi and Wariishi Hiroyuki. Gene Expression Profiling and Enzymatic Function of Phanerochaete Chrysosporium Cytochrome P450s Involved in the Metabolism of Benzo(a)pyrene. International Journal of Environmental Bioremediation & Biodegradation. 2016; 4(3):85-92. doi: 10.12691/ijebb-4-3-3

Abstract

The completed genome sequences and genomics databases currently available for plants, animals and fungi provide a scaffold for addressing the biological role of the impressive gene collection in these organisms. The white-rot fungus Phanerochaete chrysosporium encodes 149 cytochrome P450 (CYP) genes in its genome. The functions of many of these genes or their target substrates are still unknown. This study aimed at elucidating the functions of P. chrysosporium’s cytochrome P450 (PcCYP) gene repertoire using the polyclic aromatic hydrocarbon (PAH) benzo(a)pyrene (BaP) as a substrate. The set of complementary expression systems used in this study was pivotal in assigning function to the PcCYPs investigated. A cDNA microarray system targeting 133 P. chrysosporium P450 cDNAs as probes was used to investigate the differential expression patterns of PcCYPs gene diversity in response to this PAH. BaP was able to elicit a response of 12 cytochrome P450 genes strongly suggesting that it was a potential substrate of these PcCYPs. A functional analysis of the 12 PcCYP genes targeting the coding sequences of these P. chrysosporium P450 cDNAs as probes was conducted. These PcCYPs 1a, 5b, 24s, 30d, 59a, 59c and 66a were proved to be functional with a heterologous Saccharomyces cerevisiae expression system. These findings strongly suggest that species that gave a physiological response and metabolized the substrate are key to the metabolism of this PAH. This knowledge can be applied to make improved predictions on the cellular systems optimized for aromatic degradation by this fungus applicable in bioremediation.

Keywords:
phanerochaete chrysosporium benzo(a)pyrene cytochrome P450 differential expression elucidation of function

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]  Anzenbacher, P, and Anzenbacherova E. Cytochrome P450 and metabolism of xenobiotics. Cell Mol Life Sci 58:737-747. 2001.
 
[2]  Atlas, R. M. Principles of Microbiology. 2nd ed. New York: WCB/McGraw-Hill. 1997.
 
[3]  Bezalel, L., Y. Hadar, P. P. Fu, J. P. Freeman, and C. E. Cerniglia. Initial oxidation products in the metabolism of pyrene, anthracene, fluorine, and dibenzothiophenone by the white-rot fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 62: 2554-2559. 1996.
 
[4]  Bezalel, L., Y. Hadar, and C. E. Cemiglia. Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus Appl. Environ. Microbiol. 63, 2495-2501. 1997.
 
[5]  Bezalel, L., Y. Hadar, P.P. Fu, J.P. Freeman, and C.E. Cemiglia. Metabolism of Phenanthrene by the White Rot Fungus Pleurotus ostreatus. Appl. Environ. Microbiol. 62, 2547-2553. 1996.
 
[6]  Bogan, B. W., R. T. Lamar, and K. E. Hammel. Fluorene oxidation in vivo by Phanerochaete chrysosporium and in vitro during manganese peroxidase-dependent lipid peroxidation. Appl. Environ. Microbiol. 62: 1788-1792. 1996.
 
[7]  Bumpus, J. A., and S. D. Aust. Studies on the biodegradation of organopollutants by a white rot fungus. p. 404-410. In Proceedings of the International Conference for New Frontiers for Hazardous Waste Management. EPA/6009-85/025. U.S Environmental Protection Agency, Washington, D.C. 1985.
 
[8]  Bumpus, JA, Tien M, Wright D, and Aust AD . Oxidation of persistent environmental pollutants by a white-rot fungus. Science 228: 1434-1436. 1985.
 
[9]  Bumpus, J. A., and S. D. Aust. Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysosporium: involvement of the lignin degrading system. Bioessays 6: 166-170. 1987.
 
[10]  Carmichael, A. B., and L. L.Wong. Protein engineering of Bacillus megaterium CYP102: The oxidation of polycyclic aromatic hydrocarbons. Eur. J. Biochem. 268: 3117-3125. 2001.
 
[11]  Cavalieri, E.L. and E.G. Rogan. The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation. Pharmacol. Ther. 55: 183-199. 1992.
 
[12]  Cerniglia, C.E. Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation. 3: 351-368. 1992.
 
[13]  Cerniglia, C. E. and D. T. Gibson. Fungal oxidation of benzo(a)pyrene and trans 7,8- dihydroxy-7,8-dihydrobenzo(a)pyrene: evidence for the formation of a benzo(a)pyrene 7,8- diol-9,10-epoxide. J. Biol. Chem., 255, 5159-5163. 1980.
 
[14]  Cerniglia, C.E. and D.T. Gibson. Metabolism of naphthalene by Cunninghamella elegans. Appl. Environ. Microbiol. 34: 363-370. 1977.
 
[15]  Chigu, NL, Hirosue S, Nakamura C, Teramoto H, Ichinose H, Wariishi H. Cytochrome P450 monooxygenases involved in anthracene metabolism by white-rot basidiomycete Phanerochaete chrysosporium. Applied Microbiology and Biotechnology. 87. 1907-1916. 2010.
 
[16]  Collins, P. J., M. Kotterman, J. A. Field, and A. Dobson. Oxidation of anthracene and benzo (a) pyrene by laccases from Trametes versicolor. Appl. Environ. Microbiol. 62: 4563-4567. 1996.
 
[17]  Field, J. A., E. de Jong, G. Feijoo Costa, and J. A. M. de Bont. Biodegradation of polycyclic aromatic hydrocarbons by new isolates of white-rot fungi. Appl. Environ. Microbiol. 58: 2219-2226. 1992.
 
[18]  Fischer, M., M. Knoll, D. Sirim, F. Wagner, S. Funke, and J. Pleiss. The Cytochrome P450 Engineering Database: a navigation and prediction tool for the cytochrome P450 protein family. Bioinformatics. 23:2015-2017. 2007.
 
[19]  Fitzpatrick, D.A., M.E. Logue, J.E. Stajich, and G. Butler. A fungal phylogeny based on 42 complete genomes derived from supertree and combined gene analysis. BMC Evol. Biol. 6: 1471-2148. 2006.
 
[20]  Flesher, J. W., S. R. Myers, and K. H. Stansbury. The site of substitution of the methyl group in the bioalkylation of benzo(a)pyrene. Carcinogenesis. 11: 493-496. 1990.
 
[21]  Flesher, J. W., S. R. Myers, and J. W. Blake. Bioalkylation of polynuclear aromatic hydrocarbons in vivo: a predictor of of carcinogenic activity. In M. Cooke, and A.J. Dennis, (ed.), Polynuclear Aromatic Hydrocarbons: A decade of progress. Battelle Press, Columbus, OH, 261-276. 1988.
 
[22]  Ghosh, DK, Dutta D, Samanta TB, and Mishra AK. Microsomal benzo(a)pyrene hydroxylase in Aspergillus ochraceous TS: evidence for multiple forms of cytochrome P450. Biochem Biophys Res Commun. 115: 692-699. 1983.
 
[23]  Gianfreda, L., and M. A. Rao. 2004. Potential of extra-cellular enzymes in remediation of polluted soils: a review. Enzyme and Microbial Technol. 35: 339-354.
 
[24]  Guengerich, F. P. Cytochrome P450 proteins and potential utilization in biodegradation. Environ. Health Perspect. 103: 25-28. 1995.
 
[25]  Haemmerli, S. D., M. S. A Leisola, D. Sanglard, and A. Fiechter. Oxidation of benzo(a)pyrene by extracellular lignases of Phanerochaete chrysosporium: veratryl alcohol and stability of lignases. J. Biol. Chem. 261: 6900-6903. 1986.
 
[26]  Hammel, K. E., B. Kalyanaraman, and T. K. Kirk. Oxidation of polycyclic aromatic hydrocarbons and dibenzo (p) dioxins by Phanerochaete chrysosporium ligninase. J. Biol. Chem. 261: 16948-16952. 1986.
 
[27]  Hattemer-Frey, H. A. and T. C. Curtis. Benzo(a)pyrene: Environmental Partitioning and Human exposure. Toxicology and Industrial Health 7 (3):141-158. 1991.
 
[28]  Kasai, N, Ikushiro S, Hirosue S, Arisawa A, Ichinose H, Uchida Y, Wariishi H, Ohta M, Sakaki T. Atypical kinetics of cytochromes P450 catalysing 3'-hydroxylation of flavone from the white-rot fungus Phanerochaete chrysosporium. J Biochem 147: 117-125. 2010.
 
[29]  Kellner, D. G., S. A. Mavest, and S. G. Sligar. Engineering cytochrome P450s for bioremediation. Curr. Opin. Biotechnol. 8: 274-278. 1997.
 
[30]  Kim, Y.-H., Moody, J.D., Freeman, J.P., Brenzna, B., Engesser, K.-H., and Cerniglia, C.E. Evidence for the existence of PAH-quinone reductase and catechol-O-methyltransferase in Mycobacterium vanbaalenii PYR-1. J Ind Microbiol Biotechnol 31: 507-516. 2004.
 
[31]  Kirk, T. K., E. Schultz, W. J. Connors, L. F. Lorenz, and J. G. Zeikus. Influence of culture parameters on lignin metabolism by Phanerochaete chrysosporium. Arch. Microbiol. 117: 277-285. 1978.
 
[32]  Kuhn, A., H. J. Ballach, and R. Wittig. Studies in the Biodegradation of 5 PAHs (Phenanthrene, Pyrene, Fluoranthene, Chrysene und Benzo(a)pyrene) in the Presence of Rooted Poplar Cuttings. Environ. Sci. and Pollut. Res. 11: 22-32. 2004.
 
[33]  Kullman, S. W., and F. Matsumura. Metabolic pathways utilized by Phanerochaete chrysosporium for degradation of the cyclodiene pesticide endosulfan. Appl. Environ. Microbiol. 62: 593-600. 1996.
 
[34]  Kullman, S. W., and F. Matsumura. Identification of a novel cytochrome P450 gene from the white-rot fungus Phanerochaete chrysosporium, Appl. Environ. Microbiol. 63: 2741-2746. 1997.
 
[35]  Launen, L., L. Pinto, C. Wiebe, E. Kiehlmann and M. Moore. The oxidation of pyrene and benzo(a)pyrene by non-basidiomycete soil fungi. Can. J. Micobiol., 41, 477-488. 1995.
 
[36]  Lee, W, Shin SH, Hong JE, Pyo H, and Kim Y. Studies on the analysis of benzo(a)pyrene and its metabolites in biological samples by using high perfomance liquid chromatography/ fluorescence detection and gas chromatography/mass spectrometry. Bull. Korean Chem. Soc. 24:559-565. 2003.
 
[37]  Martinez, D, L.F. Larrondo, N. Putnam, M.D.Gelpke, K. Huang, J. Chapman, K.G. Helfenbein, P. Ramaiya, J.C. Detter, F. Larimer, P.M. Coutinho, B. Henrissat, R. Berka, D. Cullen, and D. Rokhsar. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat Biotechnol. 22: 695-700. 2004.
 
[38]  Masaphy, S., D. Levanon, Y. Henis, K. Venkateswarlu and S.L. Kelly. Evidence for cytochrome-P450 and P450 mediated benzo(a)pyrene hydroxylation in the white rot fungus Phanerochaete chrysosporium. FEMS Microbiol Letters. 135, 51-55. 1996.
 
[39]  Masaphy, S., D. Levanon, Y. Henis, K. Venkateswarlu and S.L. Kelly. Microsomal and cytosolic cytochrome P450 mediated benzo(a)pyrene hydroxylation in Pleurotus pulmonarius. Biotechnology Letters. 17, 969-974. 1995.
 
[40]  Murray, M., Reidy G.F. Selectivity in the inhibition of mammalian cytochromes P-450 by chemical agents. Pharmacol Rev.; 42(2): 85-101. 1990.
 
[41]  Nakamura, C. Master Thesis, Kyushu University, Japan. 2005.
 
[42]  Park, J., Park B., Jung K., Jang S., Yu K., J. Choi, S. Kong, J. Park, S. Kim, H. Kim, S. Kim, J.F. Kim, J.E. Blair, K. Lee, S. Kang, Y.H. Lee. CFGP: a web-based, comparative fungal genomics platform. Nucleic Acids Res. 36: D562-571. 2008.
 
[43]  Payne, G.A., W.C. Nierman, J.R. Wortman, B.L. Pritchard, D. Brown, R.A. Dean, D. Bhatnagar, T.E. Cleveland, M. Machida, and J. Yu. Whole genome comparison of A. flavus and A. oryzae. Med. Mycol. 44:9-11. 2006.
 
[44]  Prough, A.R, Saeki Y., and Capedevilla J. The metabolism of benzo(a)pyrene phenols by rat liver microsomal fractions. Arch..Biochem.Biophys. 122: 136 -146. 1981.
 
[45]  Sakaki, T., R. Shinkyo, T. Takita, M. Ohta, and K. Inouye. Biodegradation of polychlorinated dibenzo-p-dioxins by recombinant yeast expressing rat CYP1A subfamily. Arch. Biochem. Biophys. 401: 91-98. 2002.
 
[46]  Sambrook, J and Russel D.W. Extraction, purification, and analysis of mRNA from eukaryotic cells. In Argentine J (ed) Molecular Cloning: A Laboratory Manual, 3rd edition, volume 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor New York. 2001.
 
[47]  Shuttleworth, K.L. and C.E. Cerniglia. Environmental aspects of PAH biodegradation. Appl. Biochem. Biotechnol. 54: 291-302. 1995.
 
[48]  Van den Brink H.J.M., R. F. M. van Gorcom, C. A. M. J. J. van den Hondel and P. J. Punt. Cytochrome P450 Enzyme Systems in Fungi. Fungal Gen. Biol. 23: 1-17. 1998.
 
[49]  Wilson, S.C. and K.C. Jones. Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review. Environ. Pollut. 81: 229-249. 1993.
 
[50]  Wiseman, A. Xenobiotic-metabolising cytochromes P-450 from microorganisms. Trends Biochem. Sci. 5: 102-104. 1980.
 
[51]  Wortman, J.R., N. Fedorova, J. Crabtree, V. Joardar, R. Maiti, B.J. Haas, P. Amedeo, E. Lee, S.V. Angiuoli, B. Jiang, M.J. Anderson, D.W. Denning, O.R. White, and W.C. Nierman. Whole genome comparison of the A. fumigatus family. Med Mycol. 44:3-7. 2006.
 
[52]  http://drnelson.utmem.edu/cytochromeP450.htm.
 
[53]  Van Leeuwen, J. S., Vermeulen, N. P.E. and Chris Vos J. Yeast as a Humanized Model Organism for Biotransformation-Related Toxicity. Current Drug Metabolism, 14, 000-000. 2013.
 
[54]  Venkataramanan, S. and Yadav J. S. Role of P450 Monooxygenases in the Degradation of the Endocrine-Disrupting Chemical Nonylphenol by the White Rot Fungus Phanerochaete chrysosporium. Appl. Environ. Microbiol. 75 (17) 5570-5580. September 2009.