Genetics - Theses
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ItemCharacterisation of putative fatty acyl-CoA synthetases and genes involved in �-oxidation in Aspergillus nidulans(University of Melbourne, 2009)Aspergillus nidulans is able to use short and long chain fatty acids as sole carbon and energy sources via the 13-oxidation pathway. This pathway occurs in both peroxisomes and mitochondria of A. nidulans. While various genes encoding the mitochondrial 13- oxidation enzymes are known, the genes encoding the peroxisomal ?-oxidation proteins have not been fully described. To investigate the first step of peroxisomal 13-oxidation two putative fatty acyl-CoA dehydrogenases, AcdA and AcdB, and two putative fatty acyl-CoA oxidases, AoxA and AoxB, were identified in the genome by blast search using the Saccharomyces cerevisiae fatty acyl-CoA oxidase FoxA and a Neurospora crassa fatty acyl-CoA dehydrogenase. The localisation of these proteins, the induction of the genes and the gene deletion phenotypes have been characterised to assess their possible involvement in ?-oxidation. The results have shown that AoxA is the major fatty acyl-CoA oxidase for peroxisomal long chain fatty acid utilisation, however, the leaky loss-of-growth phenotype of the aoxA? strain implies that there are additional peroxisomal ?-oxidation pathways. The induction of the four putative fatty acyl-CoA dehydrogenases and fatty acyl-CoA oxidases in response to fatty acids was shown to be dependent on the transcriptional regulators FarA required for the induction by short and long chain fatty acids and FarB and ScfA required for short chain fatty acid induction. This pattern of induction was observed for aoxA, while acdA, acdB and aoxB show a previously undescribed induction pattern with FarB and ScfA being required for short chain and long chain fatty acid induction and FarA for short chain fatty acid induction only. The occurrence of two induction patterns implies a more complex regulation by the three regulatory proteins. Six putative fatty acyl-CoA synthetases, FatA, FatB, FatC, FatD, FaaA and FaaB, have been identified using the S. cerevisiae fatty acyl-CoA transporters and synthetases Fat1, Fat2, Faa1, Faa2, Faa3 and Faa4. Investigation of the localisation of these proteins and phenotypes associated with the deletion of these genes showed that FaaB is likely to be the major peroxisomal fatty acyl-CoA synthetase and activates fatty acids with a wide range of chain lengths. The effect of deletion of the genes encoding these putative fatty acyl-CoA synthetases on the regulation of aoxA and acuJ was investigated to determine whether any of the putative fatty acyl-CoA synthetases were required to generate activated fatty acids for induction. It is clear that there are multiple genes contributing to fatty acid utilisation and that there is considerable redundancy. Additional genome and phylogenetics analyses have identified a variety of additional proteins, which may be involved in peroxisomal ?-oxidation pathways, as well as a potential mitochondrial fatty acyl-CoA synthetase (AN4659.3). This protein is predicted to localise to mitochondria, contains a short chain fatty acyl-CoA synthetase motif and is likely to be a synthetase activating short chain fatty acids, which are utilised in mitochondria.