Molecular biological characterization and enhancement of the biodesulfurization (dsz) pathway

The focus of this research is molecular biological characterization of the biodesulfurization (dsz) pathway as found in Rhodococcus erythropolis IGTS8. These studies investigate many topics concerning the sulfur specific dsz pathway but the major focus of this research is a characterization of the dsz pathway, genes and associated components with the ultimate goal of developing a commercial biodesulfurization process. Initial worked involved the characterization of the desulfurization genes of IGTS8, but it became evident that improvement of the native Rhodococcus erythropolis IGTS8 dsz biocatalyst's activity is required to make this a commercially viable process. Research topics addressed include molecular biological enhancements of the dsz pathway genes such as engineering recombinant strains for over-expression of the dsz genes, removal of end product repression (sulfate), dsz gene expression in heterologous species and thermostabilization of the dsz genes.; This work contains a characterization of the expression of the native dsz genes in a variety of biodesulfurization conditions. The research examines the dsz genes and regulatory elements for transcriptional or translational regulation and examines the expression of the dsz genes through the kinetics of biodegradation. Mutants were isolated that are derepressed for sulfate and these cultures were compared against the native organisms. The expression of the dsz genes was monitored through the use of both molecular genetic monitoring techniques and activity specific assays. This phase of the research also included the construction of genetic manipulation tools for Rhodococcus erythropolis IGTS8, characterization of dsz operon regulation, replacement of native dsz promoter with alternative promoters, a comparison of plasmid borne dsz expression versus wild type (Rhodococcus erythropolis IGTS8), the isolation and characterization of a thermophilic dsz positive GTIS 10 and expression of the dsz operon at higher temperatures.