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Type: Thesis
Title: Disruption of carbon catabolite repression and investigation of amylase gene duplication in the filamentous fungus Aspergillus oryzae
Author: Hunter, Adrian James
Issue Date: 2018
School/Discipline: School of Biological Sciences
Abstract: Through many centuries of domestication, Aspergillus flavus, a dangerous human pathogen and producer of carcinogenic aflatoxin, has given rise to Aspergillus oryzae, a benign fungus widely cultivated in the Orient to produce traditional fermented foods and beverages. Today A. oryzae is also a source of amylases and other hydrolytic enzymes used in industry, and has been proposed to be used in an environmentally friendly treatment process that would convert the organic material in winery wastewater into protein-rich fungal biomass. Such biomass could be sold as animal feed, offsetting treatment costs and reclaiming nutrients currently being lost as waste. To enhance the efficiency of this process, carbon catabolite repression (CCR) was disrupted in A. oryzae. CCR is the repression of genes encoding enzymes for the utilisation of non-preferred carbon sources in the presence of a preferred carbon source such as glucose, which is abundant in winery wastewater. To disrupt CCR, the gene creB, encoding a deubiquitinating enzyme, was deleted in two strains of A. oryzae. In A. oryzae RIB40, creB deletion increased the production of secreted cellulases, xylanases, and amylases in inducing conditions, and greatly increased the production of secreted amylases in non-inducing and repressing conditions. Repression of amylases by glucose was much weaker in the creB-deleted strain, indicating CCR was disrupted. In contrast, deletion of creB in A. oryzae DAR3699, a strain used in soy fermentation, had no discernible effect on CCR. A. oryzae DAR3699 was shown to have weak CCR and other phenotypes characteristic of creB mutants. It was found to have a single base pair insertion in a putative micro-open reading frame in the promoter of creB, predicted to greatly reduce the efficiency of translation. Thus A. oryzae DAR3699 already possessed a loss-of-function mutation in creB. To investigate whether creB deletion would be useful for winery wastewater treatment, creB-deleted A. oryzae RIB40 and its parent were grown on synthetic winery wastewater in a bench-scale bioreactor. The two strains were found to perform similarly, with indistinguishable morphology and patterns of carbon source consumption. Although no advantage of the creB-deleted strain was observed for this application, its robust growth and increased enzyme secretion suggest it may be useful in other industrial processes. Whereas A. flavus has only one copy of the gene for alpha-amylase, various A. oryzae strains are known to have additional copies, which have presumably arisen by gene duplication during domestication. A. oryzae DAR3699 was observed to have lower amylase secretion than other strains, suggesting it might lack the additional copies. Investigation revealed that it does in fact contain a second alpha-amylase gene, but that this copy has arisen from a duplication event independent of those that produced the second and third copies in A. oryae RIB40. The duplications in the latter strain appear to have been mediated by complex transposition events involving a 9.1 kb transposable element of the Tc1/mariner class.
Advisor: Kelly, Joan
Jin, Bo
Saint, Chris
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018
Keywords: Winery wastewater
wastewater treatment
gene evolution
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