|WOLPERT, THOMAS - Oregon State University
|HYMAN, MICHAEL - North Carolina State University
|CIUFFETTI, LYNDA - Oregon State University
Submitted to: Biodegradation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2013
Publication Date: 5/14/2014
Citation: Trippe, K.M., Wolpert, T.J., Hyman, M.R., Ciuffetti, L.M. 2014. RNAi silencing of a cytochrome P450 monooxygenase disrupts the ability of a filamentous fungus, Graphium sp. to grow on short-chain gaseous alkanes and ethers. Biodegradation. 25(1):137-151. doi: 10.1007/s10532-013-9646-1.
Interpretive Summary: Some species of the soil-dwelling fungus Graphium are known to grow on unusual compounds including propane and butane. These same species are also able to grow on environmental pollutants like the solvent tetrahydrofuran (THF). After Graphium grows on propane or THF, it degrades other pollutants, including methyl tert butyl ether (MTBE) and 1,4-dioxane. Very little information is known about the unique mechanism that allows Graphium to grow on or degrade these unusual chemicals. In this paper, we identified the protein that converts propane, THF, MTBE, 1-4-dioxane into more palatable compounds that can be readily consumed by the fungus. Because the dirivatives of these compounds are commonly used as growth substrates by many organisms, the identification of this unique protein, which belongs to the cytochrome P450 monooxygenase family, provides knowledge about the biochemical step that allows Graphium to occupy this strange ecological niche. Understanding the biochemical pathway that allows Graphium to grow on pollutants not only contributes to our knowlegde of fungal biochemistry, but also provides insight and technology for cleaning up toxic compounds from soils and groundwater.
Technical Abstract: Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was identified, cloned and functionally characterized as an alkane-oxidizing cytochrome P450 (GSPALK1). Analysis of CYP52L1 suggests that it is a member of the CYP52 cytochrome P450 family, which is comprised of medium- and long-chain alkane-oxidizing enzymes found in yeasts. However, phylogenetic analysis of GSPALK1 with other CYP52 members suggests they are not closely related. Post-transcriptional ds-RNA-mediated gene silencing of CYP52L1 severely reduced the ability of this fungus to oxidize alkanes and ethers, however, downstream metabolic steps in these pathways were unaffected. Collectively, the results of this study suggest that GSPALK1 is the enzyme that catalyzes the initial oxidation of alkanes and ethers but is not involved in the later steps of alkane or ether metabolism.