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.
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