Between Dec 19, 2024 and Jan 2, 2025, datasets can be submitted to DRUM but will not be processed until after the break. Staff will not be available to answer email during this period, and will not be able to provide DOIs until after Jan 2. If you are in need of a DOI during this period, consider Dryad or OpenICPSR. Submission responses to the UDC may also be delayed during this time.

Browsing by Author "Frias, Janice Alina"

Now showing 1 - 1 of 1
  • Thumbnail Image
    Item
    Microbial synthesis of fuel hydrocarbons: enzymes and metabolic pathways.
    (2011-01) Frias, Janice Alina
    Petroleum is the major source of motor fuels and commodity chemicals in modern society. Petroleum consists of hydrocarbons and is formed largely by abiotic reactions. Hydrocarbons are also biosynthesized by plants, insects, and microbes. Therefore, understanding how hydrocarbons are biosynthesized could lead to a new source of fuels and chemicals. Biohydrocarbons would be superior to ethanol and biodiesel in many ways. To produce biohydrocarbons on large scale, it is necessary to study the genes and enzymes involved in the synthesis of hydrocarbon molecules. This thesis research deals with microorganisms that synthesize alkanes or alkenes. The bacterium Vibrio furnissii M1 was reported to produce considerable quantities of diesel-length alkanes. We obtained the bacterium from the institute in Japan where it was studied. Our studies reached the conclusion that the strain did not produce alkanes. Subsequent research focused on Micrococcus luteus ISU and Stenotrophomonas maltophilia that were reported to produce C25-C31 alkenes. Previous studies indicated that the alkenes derived from fatty acids joined at or near the carboxyl carbon atoms in a process denoted as a head-to-head condensation mechanism. The genes, enzymes, and metabolic intermediates in the head-to-head biosynthetic pathway were not identified in those studies. My research showed that some Arthrobacter strains produced head-to-head hydrocarbons and others did not. A putative hydrocarbon biosynthetic gene cluster was identified in the genomes of the strains capable of making hydrocarbons. Research done in Shewanella, another organism identified and verified to produce hydrocarbons, verified experimentally that the gene cluster was indeed involved in long-chain alkene synthesis. The four genes identified, oleABCD, encode for proteins from the following superfamilies: thiolase, -hydrolase, AMP-dependent ligase/synthase, and short chain dehydrogenase, respectively. The OleC protein from Stenotrophomonas maltophilia was expressed in Escherichia. coli and was purified to homogeneity. It was shown to react with ATP and activity increased in the presence of long chain -hydroxy acids. The OleC enzyme was crystallized in the presence of 5’-AMP and synchotron diffraction data collected to 3.4 Angstrom resolution. There was significant mobility in the linker region between the N terminal domain and the C terminal domain making detailed structure elucidation impossible. OleA was thought to catalyze the crucial first step in the biosynthetic pathway and thus provided the key to reconstituting alkene biosynthesis in vivo. The Arthrobacter OleA was not active in vitro. Subsequently six synthetic oleA genes were cloned into E. coli expression hosts. The Xanthamonas campestris OleA protein expressed well in E. coli and comprised approximately 50% of the soluble protein. The his-tagged protein was purified to homogeneity in one-step via Ni-column chromatography. OleA has a subunit MW of 38,800 Da and a subunit stoichiometry of 1.75. OleA was assayed with fatty acyl-CoA substrates by measuring the release of coenzyme A (CoA) using 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB). Surprisingly, the ratio of CoA released:fatty-acyl group consumed was 1:1 whereas homologous biosynthetic thiolases show a ratio of 1:2. OleA was indicated to catalyze a Claisen condensation of fatty acyl groups coupled with hydrolysis of the generated CoA ester to produce a -keto acid intermediate. The -keto acid intermediate was rigorously identified by high pressure liquid chromatography and mass spectrometry of the methyl ester. Details of the alkene biosynthetic pathway remain to be elucidated. This thesis describes research in which two of the four enzymes have been purified to homogeneity, details of their reactions characterized, the proteins were crystallized, and details of protein structure were revealed. The OleA, OleC, and OleD proteins were combined in vitro and shown to produce alkenes. Moreover, the product of the OleA reaction produced synthetically was shown to be a competent intermediate for alkene synthesis by OleC and OleD. The OleB protein is not required and its role in alkene biosynthesis remains to be elucidated. {NOTE THAT CHEMICAL SYMBOLS WERE NOT COPIED PROPERLY SEE PDF ABSTRACT}