Smith, Megan2021-10-132021-10-132021-08https://hdl.handle.net/11299/224968University of Minnesota Ph.D. dissertation. 2021. Major: Microbiology, Immunology and Cancer Biology. Advisors: Lawrence Wackett, Jake Bailey. 1 computer file (PDF); xiv, 172 pages.Various microorganisms posses the ability to create long chain olefinic hydrocarbons, which can be useful as biofuel precursors or for the creation of specialty chemicals. Additionally, the biosynthesis pathways for the production of these olefins can be used to create other high value compounds such as β-lactone therapeutics and surfactants. Our lab has identified hundreds of possible olefin and β-lactone biosynthetic gene clusters, however many of the end products are unknown. In order to test the diversity of olefins produced, we focused our efforts on the first enzyme in the pathway, OleA. OleA is a thiolase that condenses two fatty acyl-CoAs into a β-keto acid, which eventually becomes the olefin or β-lactone end-product. Earlier research has shown that OleA is responsible for the overall shape of the end-product, through its substrate specificity. Thus, we surmised that studying OleA would provide insight on the natural variance of Olefin and β-lactone compounds produced in nature. Through a collaboration with the Joint Genome Institute we have sampled a total of 72 OleA recombinant proteins using a new colorimetric assay that was developed through the use of inexpensive and readily available p-nitrophenyl compounds as substrate analogs. Our research has shown that many of these OleA proteins are able to accept a wide variety of compounds, many of which are not found in nature. In addition to the development of the screen, we discovered that the p-nitrophenyl ester could only participate as the first substrate of the Claisen condensation reaction of OleA. We then exploited this to produce the β-keto acid product using p-nitrophenyl esters, as well as further refine the OleA mechanism, by demonstrating the directionality of the Claisen condensation. Finally, we showed the addition of cetyltrimethylammonium bromide (CTAB), below critical micelle concentrations, can improve the yield of OleA, both when using native or alternative substrates. This improves the potential for OleA to become a biotechnologically important enzyme, and points to future ways we can improve the overall enzyme pathway to generate large amounts of desired product. This thesis represents a first step in a larger goal of producing industrially relevant β-lactone compounds and their derivatives.enB-lactoneCoAOleAOlefinp-nitrophenolThiolaseThesis or Dissertation