Browsing by Subject "Ketones"
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Item Hydrocarbon biosynthesis by bacteria : genes and hydrocarbon products.(2010-12) Sukovich, David JohnVarious 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.Item The Role of Hepatocyte D-Β-Hydroxybutyrate Dehydrogenase In Ketone Body Metabolism And Liver Health(2021-09) Stagg, DavidThroughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. This thesis seeks to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, dietary-induced mouse models of nonalcoholic fatty liver disease (NAFLD), and complementary physiological approaches in novel cell type-specific knockout mice, the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies, are quantified. Exogenously administered AcAc is reduced to D-βOHB, and increases hepatic NAD+/NADH ratio, reflecting hepatic BDH1 activity. Livers of hepatocyte-specific BDH1 deficient mice produced no D-βOHB, but due to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte specific BDH1 deficient mice, maintained on either a chow or NAFLD-inducing western-style diet, showed diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis but normal overall hepatic energy charge. Furthermore, the livers of knockout mice maintained on a 60% high fat diet were less fibrotic, with reduced markers of oxidative stress, than littermate controls. Collectively, this thesis illustrates how ketone bodies and BDH1 activity influence liver homeostasis and health. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production, with a beneficial effect on liver fibrosis. Therefore, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis, and augmentation of hepatic BDH1 activity could prove beneficial in the treatment of NAFLD.