With the increasing demand of upgrading the quality of petroleum, optimizing the efficiency of biodesulfurization (BDS) is necessary. R.... Show moreWith the increasing demand of upgrading the quality of petroleum, optimizing the efficiency of biodesulfurization (BDS) is necessary. R. erythropolis 5F [pRESX dszABC] encodes the enzymes of the 4S pathway (on the dszABC operon) which catalyze the conversion of dibenzothiophene (DBT) to hydroxybiphenyl (2-HBP) plus sulfite. Our research attempts to increase the nutritional requirement of the cell for sulfur and to use selective pressure to obtain improved desulfurization. The genetic engineering strategy involves inserting the sulpeptide 1 (S1) gene, which encodes a sulfur-rich polypeptide, between dszA and dszBC in the dszABC operon to increase sulfur demand and force the strain into a spiral of ever increasing dszABC expression and thus desulfurization ability. Insertion of the chloramphenicol resistance (Cm) gene between dszAS1 and dszBC provides an additional method to select strains with higher desulfurization ability by their increasing resistance to chloramphenicol. We completed directed evolution for 22 passages for all three strains (those containing pRESX dszABC, pRESX dszAS1BC, or pRESX dszAS1CmBC transformed into strain 5F) and measured the desulfurization activity at selected passages. The results show that directed evolution did improve the desulfurization abilities in the first ten passages in the strains expressing dszABC and dszAS1CmBC, but reduced the desulfurization ability in the strain expressing dszAS1BC. For all three strains and all passages tested, the highest desulfurization activity was 83.6 μmol DBT/g DCW/hour in the dszABC bearing strain. Thus, the presence of S1 had a negative effect on the rate of desulfurization. A possible explanation for these results is that mutations occurring during the selection process enhance the efficiency of sulfur utilization and S1 alone can thus not force the strains to improve desulfurization. M.S. in Biology, December 2013 Show less
