Metabolic engineering of a dibenzothiophene biodesulfurization operon

Sulfur oxides produced from the combustion of fossil fuels are detrimental to the environment and human health. The level of sulfur in gasoline and diesel is regulated to limit the amount of sulfur released by automobiles and trucks. These levels are being lowered due to environmental and health concerns, and the current method is costly and inefficient when applied to these new goals. One possible alternative to the current hydrodesulfurization method is the use of microorganisms to remove the sulfur. This process, biodesulfurization, requires much milder processing conditions, gives higher specificity, and it does not require hydrogen.; Previous studies have determined that a bacterium, Rhodococcus erythropolis IGTS8, could convert dibenzothiophene to hydroxybiphenyl. The three genes required for this transformation were cloned and the pathway was elucidated. It was shown that the first three steps in the process require reduced flavin, and that in vivo production of a heterologous flavin reductase can increase the speed of the initial desulfurization steps. However, the overall rate of sulfur removal is limited by the last step in the pathway, catalyzed by the enzyme DszB.; The goal of the present work was to increase the rate of desulfurization by increasing the production of DszB. It was found that modifications to the region immediately upstream designed to increase DszB production often had the opposite result. A series of green fluorescence protein and desulfurization protein fusions were created to facilitate the generation of a plasmid with increased DszB production. Randomized upstream sequences and fluorescence screening were used to generate and isolate a sequence that produced a 9-fold higher rate of desulfurization.