Microbial detoxification of pollutant chemical is an effective way to reduce human health hazards from contaminated environments. Phamerochaete chrysosporium, a ligninolytic white rot fungus has been used in bioremediation under nutrient-limited (ligninolytic) environments, based on expression of its exo-peroxidases triggered under these conditions. However, there is increasing evidence that P450 monooxygenation initiates xenobiotic catabolism under nutrient-rich conditions when exo-peroxidases are suppressed, and that P450 reaction products produced subsequently may link up with the ligninolytic pathways as the primary nutrients are depleted. The long-term objective of this research is to better understand the relationship of catabolic P450 systems to the overall pathways of xenobiotic detoxification in white rot fungi and for development of enzyme-based or whole cell-based technologies for bioremediation.
The specific aims are to (i) complete the isolation and characterization of the gene for a catabolic P450 monooxygenase, (ii) study its xenobiotic induction and regulation under varied physiological conditions; (iii) manipulate its expression in heterologous hosts and the native host in conjunction with the recently characterized P450 reductase gene; (iv) study its catalytic role in vivo and in vitro; and (v) evaluate the practical application in bioremediation using optimized P450 conditions and engineered strains. A partial sequence of the P450 gene is available and will be used in screening newly constructed genomic and cDNA libraries for gene isolation. Xenobiotic induction and regulatory conditions will be examined based on mRNA and enzyme levels. The monooxygenase cDNA will be coexpressed with the available reductase cDNA is Saccharomyces cerevisiae for whole cell-based and microsome-based catalytic assays or expressed in a baculovirus system for enzyme production for in vitro assays. Enzyme substrate(s) will be identified based on induction results, the use of P450 null mutant haploids and gene amplified strains of P. chrysosporium constructed for this purpose, and/or in vitro assays using expressed enzymes. Optimized P450 conditions and strains will be tested in bench top models of bioremediation.
|Bhattacharya, Sukanta S; Syed, Khajamohiddin; Shann, Jodi et al. (2013) A novel P450-initiated biphasic process for sustainable biodegradation of benzo[a]pyrene in soil under nutrient-sufficient conditions by the white rot fungus Phanerochaete chrysosporium. J Hazard Mater 261:675-83|
|Syed, Khajamohiddin; Porollo, Aleksey; Miller, David et al. (2013) Rational engineering of the fungal P450 monooxygenase CYP5136A3 to improve its oxidizing activity toward polycyclic aromatic hydrocarbons. Protein Eng Des Sel 26:553-7|
|Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai et al. (2013) CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes. Appl Environ Microbiol 79:2692-702|
|Syed, Khajamohiddin; Yadav, Jagjit S (2012) P450 monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium. Crit Rev Microbiol 38:339-63|
|Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai et al. (2011) A fungal P450 (CYP5136A3) capable of oxidizing polycyclic aromatic hydrocarbons and endocrine disrupting alkylphenols: role of Trp(129) and Leu(324). PLoS One 6:e28286|
|Syed, Khajamohiddin; Kattamuri, Chandramohan; Thompson, Thomas B et al. (2011) Cytochrome b? reductase-cytochrome b? as an active P450 redox enzyme system in Phanerochaete chrysosporium: atypical properties and in vivo evidence of electron transfer capability to CYP63A2. Arch Biochem Biophys 509:26-32|
|Syed, Khajamohiddin; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan et al. (2010) Genome-to-function characterization of novel fungal P450 monooxygenases oxidizing polycyclic aromatic hydrocarbons (PAHs). Biochem Biophys Res Commun 399:492-7|
|Subramanian, Venkataramanan; Doddapaneni, Harshavardhan; Syed, Khajamohiddin et al. (2010) P450 redox enzymes in the white rot fungus Phanerochaete chrysosporium: gene transcription, heterologous expression, and activity analysis on the purified proteins. Curr Microbiol 61:306-14|
|Subramanian, Venkataramanan; Yadav, Jagjit S (2009) Role of P450 monooxygenases in the degradation of the endocrine-disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium. Appl Environ Microbiol 75:5570-80|
|Subramanian, Venkataramanan; Yadav, Jagjit S (2008) REGULATION AND HETEROLOGOUS EXPRESSION OF P450 ENZYME SYSTEM COMPONENTS OF THE WHITE ROT FUNGUS PHANEROCHAETE CHRYSOSPORIUM. Enzyme Microb Technol 43:205-213|
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