Dibenzothiophene (DBT) is an organosulfur compound found in petroleum that is refractory to the current hydrodesulfurization (HDS) method in... Show moreDibenzothiophene (DBT) is an organosulfur compound found in petroleum that is refractory to the current hydrodesulfurization (HDS) method in refineries and raises the need for alternative methods such as biodesulfurization. Rhodococcus erythropolis strain IGTS8 naturally contains a dszABC operon, which encodes enzymes that can desulfurize DBT through the 4S pathway. Desulfurization-negative Rhodococcus opacus was transformed with plasmids pRESXdszABC and pRESXdszAS1BC, conferring the ability to desulfurize DBT. pRESXdszAS1BC had been modified from pRESXdszABC by placing a synthetic “sulpeptide gene” (S1) within the operon between dszA and dszB; S1 encodes a short polypeptide with high methionine and cysteine content, which puts additional sulfur demands on the 4S pathway when DBT is the only source of sulfur. Here we performed directed evolution on the two transformed R. opacus strains for 50 passages in a minimal (CDM) medium with DBT as the sole sulfur source in an attempt to drive evolution of greater DBT desulfurization activity. Desulfurization specific activity experiments were performed every 4 to 10 passages to compare the specific activities of these strains through the production of 2-HBP as measured by the Gibbs assay. Desulfurization positive strain Rhodococcus erythropolis IGTS8 was used as the control culture. R. opacus/pRESXdszABC demonstrated a maximum increase of 28-fold in desulfurization specific activity after 40 passages to a level as high as that of the control culture R. erythropolis IGTS8. Thereafter (passages 41-50), there was a 7% decrease from the maximum of desulfurization activity level (passage 40). R. opacus/pRESXdszAS1BC, however, showed only a maximum increase of 4-fold in specific activity after 37 passages. Moreover, there was a 61% decrease from the ix maximum of desulfurization activity level (passage 37) thereafter (passage 38-50). These could be due to the unexpected mutations and/or epigenetic changes in the pRESXdszAS1BC plasmid or host genomic sequences. Further DNA sequence analysis will be helpful in identification of these possible mutations. M.S. in Biology, December 2013 Show less
