Sukovich, David John2011-03-022011-03-022010-12https://hdl.handle.net/11299/101046University of Minnesota Ph.D. dissertation. December 2010. Major: Microbiology, Immunology and Cancer Biology. 1 computer file (PDF); xvi, 190 pages, appendices A-F. Ill. (some col.)Various publications have reported that microorganisms have the ability to produce hydrocarbons. One of these organisms, Vibrio furnissii M1, was reported to produce n-alkanes. Genomic analysis and biochemical studies revealed that the findings reported by Park et al. were not reproducible in our laboratory. Other heterotrophic bacteria were shown to produce hydrocarbons though. One of these organisms, Shewanella oneidensis MR-1, was found to produce 3,6,9,12,15,19,22,25,28-hentriacontanonaene. Hydrocarbon production in S. oneidensis was dependent upon the polyunsaturated fatty acid synthesis pathway and a relationship between temperature and hydrocarbon production was identified. Genomic analysis and mutation studies found that hydrocarbon production was dependent upon a gene cluster, designated oleA, oleB, oleC, and oleD. The OleA protein condenses two fatty acyl CoA chains in a head-to-head manner to produce a compound that, if the OleBCD proteins are not present, is spontaneously decarboxylated to a ketone. Homologs to the oleABCD genes were found in all heterotrophic bacteria reported to produce hydrocarbons. Searches of genomic databases found that 1.9% of all sequenced genomes have oleABCD gene homologs. These bacteria include members from the γ- and δ-Proteobacteria, Actinobacteria, Verrucomicrobia, Planctomycetes, and Chloroflexi Phyla. Bacteria containing the oleABCD homologs not previously characterized for hydrocarbons were obtained and tested for polyolefin production. It was found that if the genes were present, bacteria produced alkenes. A correlation between OleA amino acid sequence and product formation was also discovered. When different OleA proteins were expressed heterologously in non-native bacterial backgrounds, the bacteria hosts were able to produce ketones. Ketone production could be increased using alternative plasmid promoters and regulation sequences. Preliminary experiments investigated strategies for cloning and expressing an oleA gene in cyanobacteria heterologously. Also, exploratory experiments were conducted to determine if ketone production by Shewanella might enhance cell growth when antibiotics, detergents, or other potentially inhibitory chemicals were added to the growth media.en-USHydrocarbonsKetonesOleMicrobiology, Immunology and Cancer BiologyThesis or Dissertation