|Institution:||Illinois Institute of Technology|
|Keywords:||PH.D in Biology, December 2013|
|Full text PDF:||http://hdl.handle.net/10560/3271|
Rhodococcus baikonurensis CW25 was transformed with the Rhodococcus erythropolis strain IGTS8 desulfurization operon (dszABC, which encodes the enzymes of the “4S” desulfurization pathway) or this operon modified to contain a synthetic cysteine-methionine rich “sulpeptide” gene (S1) (dszAS1BC). The two CW25 derivatives were subjected to directed evolution to select faster growing cells using the key 4S pathway substrate dibenzothiophene (DBT) as the sole source of sulfur. Data of cell doubling times verified the success of selection of cultures with increasingly rapid growth. The desulfurization activities of resting cells of early passages demonstrated improvements, and the highest activity of the dszAS1BC-bearing CW25 derivative was 115% higher than that of the CW25 derivative without S1. In addition, a trend of initial high activity was followed by a decrease in subsequent passages. Rates of DBT metabolism of growing cells demonstrated a different trend, probably because the activity of growing cells concurrently reflects the activity of DszABC enzymes and the growth rates of the recombinants. Dry cell weights fluctuated during the evolution process, probably because of variations in the efficiency of the conversion of the sulfur in DBT into sulfite, then into sulfate or biomass, or, for the S1-bearing cells, because the secretion of the S1 peptide from cells might have variable efficiency. A mixed culture of two Paenibacillus species (“W” and “Y”) was isolated that can metabolize DBT at temperatures up to 54 ºC. Strain Y is the only one of the two with desulfurization activity, while strain W enhances the desulfurization ability of Y. The W-Y culture may be a useful starting point for selection of desulfurization cultures with even greater thermal stability. xiii Ethanologenic Escherichia coli strain FBR5 was compared with Vitreoscilla hemoglobin (VHb)-expressing FBR5 (TS3) regarding the concentrations of ATP, NAD+, NADH, NAD+/NADH ratio; and growth and ethanol production at various points during growth. The significant finding was that the NAD+/NADH ratio for TS3 was lower in early growth, but higher in later growth compared to that for FBR5. This is probably because more NADH was required by TS3 for its enhanced ethanol production and VHb-related increased respiration under microaeration conditions.