Lignin is the second most abundant renewable organic polymer in the biosphere. Lignin biodegradation is of utmost importance in the earth's carbon cycle not only because of its great abundance but also because of its recalcitrance. Phanerochaete chrysosporium, a filamentous ligninolytic basidiomycete, produces H202-generating glucose oxidase and H202 dependent extracellular heme peroxidases called ligninases, but only during idioptase (secondary metabolism). Ligninases constitute a class of six or more related proteins that appear to be encoded by different genes. There has been much recent interest in ligninases because these proteins, in addition to their involvement in degradation of lignin, also play a key role in the detoxification of recalcitrant environmental pollutants such as dioxins, DDT, lindane and benzopyrenes, which constitute a serious public health hazard. Our long term objective is to isolate and characterize various lignin degradation genes from P. chrysosporium and study the molecular organization, expression and regulation of these genes. In this study, we propose to characterize ligninase genomic clones including construction of detailed restriction maps, determine the presence and nature of introns, determination of gene boundaries, and DNA sequence determination of the 5' upstream region (approximately 1 kb) and the entire coding region. Upstream regulatory regions will be identified using DNase I protection analysis. Transcription specific binding proteins, if any present, will be isolated and characterized. Furthermore, chromosome organization of P. chrysosporium and distribution of ligninase genes in the chromosome(s) will be determined. In this study, we also propose to isolate and characterize cDNA and genomic clones for glucose oxidase, another enzyme expressed during secondary metabolism and is reported to be associated with the lignin degradation system. The cDNA and genomic clones for glucose oxidase will be characterized the same way as proposed for the ligninase clones. The results of the proposed research will not only aid in the understanding of gene structure and organization in P. chrysosporium but also will make an important fundamental contribution to the knowledge base on the biology and genetics of filamentous fungi. The data obtained will additionally be useful in producing genetically engineered strains of P. chrysosporium that give high yields of ligninase, an enzyme with many important practical applications.
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