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dc.creatorSun, Kaiwen
dc.date.accessioned2020-09-14T18:02:46Z
dc.date.available2020-09-14T18:02:46Z
dc.date.created2020-10
dc.date.issued2020-09-14
dc.date.submittedOctober 2020
dc.identifier.urihttp://hdl.handle.net/10388/13012
dc.description.abstractVery long chain polyunsaturated fatty acids (VLCPUFAs), such as docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:5-5,8,11,14,17), are essential for humans and animals since they are parts of the cell membrane and are involved in mediating various physiological processes. Thraustochytrium sp. ATCC 26185 is a marine protist that can produce large amounts of VLCPUFAs such as DHA and DPA (docosapentaenoic acid, 22:5-4,7,10,13,16) for human food and animal feed. Biosynthesis of these fatty acids in Thraustochytrium is catalyzed by a polyunsaturated fatty acid (PUFA) synthase that comprises three subunits, each with multiple catalytic domains. Three dehydratases (DH) domains in the PUFA synthase are believed to be responsible for coordinately introducing multiple double bonds in VLCPUFAs; however, the exact function of these domains remains to be determined. In this research, two DH domains (DH1 and DH2) in subunit-C of the PUFA synthase that have sequence similarity to E. coli FabA were functionally analyzed by site-directed mutagenesis and domain deletion analyses. Site-directed mutagenesis analysis showed that mutation of a histidine residue at calalytic site into alanine in DH1 of the PUFA synthase resulted in the complete loss of activity in the biosynthesis of all VLCPUFAs. Mutation of catalytic residue histidine into alanine in DH2 resulted in the production of a small amount of DPA, but not DHA. In addition, the deletion of DH1 domain also led to the complete loss of function while deletion of DH2 domain resulted in the production of only a very small amount of DPA. These results indicate that two FabA-like domains of the PUFA synthase possess distinct functions. DH1 domain, but not DH2 domain, is essential for the biosynthesis of VLCPUFAs, and DH2 domain is required for the biosynthesis of DHA. The PUFA synthase must have both for the efficient production of VLCPUFAs. Next, expression and purification of the PUFA synthase were attempted for future structure analysis. Partial purification of these subunits was accomplished using a His-tagged protein purification system and verified with western blot analysis. Successful purification of the subunit of the PUFA synthase expressed in E. coli would be an important step forwards for studying the structure and activity of each subunits of this enzyme and offer strategies for elucidating the molecular mechanism for the biosynthesis of VLCPUFAs.
dc.format.mimetypeapplication/pdf
dc.subjectPolyunsaturated fatty acid Synthase, Very long chain polyunsaturated fatty acids, Thraustochytrium sp. ATCC 26185, dehydratases (DH) domains, E. coli FabA
dc.titleFunctional Analysis of Dehydratase Domains of a Polyunsaturated fatty acid Synthase from Thraustochytrium sp. by Mutagenesis
dc.typeThesis
dc.date.updated2020-09-14T18:02:46Z
thesis.degree.departmentFood and Bioproduct Sciences
thesis.degree.disciplineApplied Microbiology
thesis.degree.grantorUniversity of Saskatchewan
thesis.degree.levelMasters
thesis.degree.nameMaster of Science (M.Sc.)
dc.type.materialtext
dc.contributor.committeeMemberNapper, Scott
dc.contributor.committeeMemberTanaka, Takuji
dc.contributor.committeeMemberQiu, Xiao
dc.contributor.committeeMemberLuo, Yu
dc.creator.orcid0000-0002-3057-0499


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