Studies on the Molecular Cloning of "Aryletherase" From a Ligninolytic Erwinia Sp.: Enhancement of Gene Expression.
Date of Award
Doctor of Philosophy (PhD)
Recombinant DNA techniques were used to enhance the expression of a gene encoding "aryletherase" activity. This gene, apparently lacking regulatory elements, was present on a 792bp BamHI DNA fragment of Erwinia sp. genomic DNA, that was cloned into Escherichia coli using the plasmid vector pBR322. To obtain a larger fragment of Erwinia genomic DNA carrying the "aryletherase" gene and its regulatory regions, an Erwinia sp. genomic DNA library was constructed in the lambda phage EMBL3. Screening the library with nick translated 792bp BamHI fragment identified several recombinant phage which demonstrated homology with the probe. Restriction digestion of DNA from one of these phage tentatively localized the 792bp BamHI fragment within a 3.1kb SmaI fragment. Restriction endonuclease mapping of the 3.1kb SmaI fragment, subcloned into pUC19, confirmed the presence of the 792bp fragment with flanking regions of Erwinia genomic DNA. E. coli transformants harboring the recombinant plasmids pJC39 or pJC2 stably expressed etherase activity which was assayed by the release of p-nitrophenol from p-nitrophenyl-(beta)-D-glucoside (PNPG). Transformants also degraded the lignin model compound vanillin by a mechanism thought to involve demethoxylation via ether cleavage. Tn5 mutagenesis localized the putative etherase gene within a 2100bp BamHI-SmaI region of the 3.1kb insert of pJC39.
Cary, Jeffrey William, "Studies on the Molecular Cloning of "Aryletherase" From a Ligninolytic Erwinia Sp.: Enhancement of Gene Expression." (1986). LSU Historical Dissertations and Theses. 4287.