The long-term objective of this project is to acquire new molecular genetic understanding of microbial metabolism of use in collaborative research on the development of improved biotreatment technologies. Targeted compounds are environmentally recalcitrant polyaromatic hydrocarbons and N-heterocyclics (PAH/ NHA), and sulfonated azo dyes. The emphasis is upon aerobic metabolism via cytochrome P450 monooxygenation for the first category, and via oxygen insensitive azo bond cleavage of the azo dyes. Fungal and/or bacterial microorganisms known to carry out these detoxication steps are available, including several that were isolated in other projects of this program. P450 genes will be isolated from microbes processing important monooxygenases and characterized for their enzyme products, by sequence modification and expression in Escherichia coli. Relatively few microbial P450s have been genetically characterized but the available evidence indicates the existence of a large number of highly divergent isomers, present as only a few isomers per microbe. For isolation of P450 genes we propose a possibly general method that targets small segments in the most conserved region of this highly sequence divergent gene superfamily.
Specific Aims 1 and 2: I test two related PCR strategies using the filamentous fungus Cunninghamella elegans and soil mycobacterium strains respectively, for the isolation of P450 genes of PAH/N-heterocyclics metabolism;
Specific Aim 3 : Isolate P450 genes based upon P450 enzymes purified from Cunninghamella sp, as an alternative to Aim 1:
Specific Aim 4 : Isolate and characterize oxygen insensitive azo reductase genes from aerobic Gram negative rod C7. Previously purified enzyme to develop a gene probe;
Specific Aim 5 : Prepare and test pathway using specific gene probes for collaborative studies of microbial communities in biodegradation systems.
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