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| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | Farah Diba Abu Bakar,Dr. | |
| dc.contributor.author | Selina Oh Siew Ling (P38420) | |
| dc.date.accessioned | 2023-10-13T09:43:32Z | - |
| dc.date.available | 2023-10-13T09:43:32Z | - |
| dc.date.issued | 2012-09-18 | |
| dc.identifier.other | ukmvital:120079 | |
| dc.identifier.uri | https://ptsldigital.ukm.my/jspui/handle/123456789/500444 | - |
| dc.description | Trichoderma and Aspergillus have been extensively used as model organisms for diverse transformation and expression systems. The aim of this study is to produce new fungal expression systems using local filamentous fungi as alternatives to those covered by patents. The approach involves the construction of expression vectors compatible with selected hosts, establishment of efficient protoplasting and transformation protocols and validation of the fungal expression systems through the expression of reporter genes. Development of an expression system employing T. virens and A. oryzae was based on a hygromycin antibiotic selection and a pyrG auxotrophic marker, respectively. The construction of the expression vectors for T. virens was carried out by assembling the expression cassettes, antibiotic resistance selection marker cassette (hygromycin cassette) and T. virens rDNA fragment into pUC19. Transformation of the vectors was successfully achieved whereby the positive transformants of T. virens showed resistance towards hygromycin and integration at the rDNA loci. Subsequently, the heterologous reporter protein of the T. virens expression system which is the Glomerella cingulata cutinase was observed to be expressed and secreted into the medium, however, in low amounts. For the development of the A. oryzae strain S1 expression system, in turn, a uridine auxotroph was isolated by deleting the pyrG gene using a targeted gene replacement approach. An episomal plasmid (ANEp2) carrying a heterologous β-galactosidase reporter gene was used to functionally test for pyrG complementation and study the strength of selected promoters in the A. oryzae pyrG auxotrophic mutant. The effectiveness of the ANEp2 plasmid in pyrG complementation and β-galactosidase expression using the A. oryzae pyrG mutant as host, were determined. ANEp2-transformed A. oryzae pyrG mutant showed very high levels of β-galactosidase activity as compared to the untransformed strain. In this study, constitutive promoters that are, the glyceraldehyde-3-phosphate dehydrogenase gene (GPD1) from T. virens and translation elongation factor 1 (TEF1) gene from A. oryzae; and inducible gene promoters that are the NAG1 (chitobiase gene) from T. virens and glucoamylase A (glaA) promoter from A. oryzae, were analysed. The development of an A. oryzae strain S1 expression system based on an auxotrophic selection marker (pyrG) using promoter-based ANEp2 variants was successful whereby the reporter protein LacA was overexpressed showing strongest expression by the A. oryzae TEF1 promoter that was comparable with the glaA promoter from A. niger. In addition via an enzyme assay, the A. oryzae strain GR6 pyrGΔ0 was also shown to overexpress the heterologous cutinase. Thus, it can be concluded that the A. oryzae expression system emerged as the more promising system as compared to the T. virens expression system for heterologous protein production based on the target protein production yield and the efficiency of the molecular expression toolboxes,Certification of Master's/Doctoral Thesis" is not available | |
| dc.language.iso | eng | |
| dc.publisher | UKM, Bangi | |
| dc.relation | Faculty of Science and Technology / Fakulti Sains dan Teknologi | |
| dc.rights | UKM | |
| dc.subject | Universiti Kebangsaan Malaysia -- Dissertations | |
| dc.subject | Dissertations, Academic -- Malaysia | |
| dc.subject | Trichoderma | |
| dc.subject | Aspergillus | |
| dc.subject | Filamentous fungi | |
| dc.title | Development of the filamentous fungi, Trichoderma and Aspergillus expression system for heterologous protein production | |
| dc.type | Theses | |
| dc.format.pages | 239 | |
| dc.identifier.callno | QK625.M7O377 | |
| dc.identifier.barcode | 002759(2012) | |
| Appears in Collections: | Faculty of Science and Technology / Fakulti Sains dan Teknologi | |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| ukmvital_120079+SOURCE1+SOURCE1.0.PDF Restricted Access | 1.35 MB | Adobe PDF |  View/Open |
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