AbstractsBiology & Animal Science

"This thesis is presented in fulfilment of the requirements for the degree of Doctor of Philosophy". Bibliography: pages 206-240. 1. Introduction  – 2. Materials and methods  – 3. Expression of fluorescent proteins GFP2 and VenusYFP in Trichoderma reesei  – 4. Optimisation of sample preparations for microscopy studies  – 5. Optimisation of the culture medium - the effect of initial medium pH on the expression of the CBHI-Venus fusion protein  – 6. Visualisation of the major organelles in secretory pathway of the filamentous fungus Trichoderma reesei  – 7. Tracking secretion of the cellobiohydrolase I (CBHI)-Venus fusion protein through the hyphae of Trichoderma reesei  – 8. Visualisation of protein interactions between BiP1 and CBHI in T. reesei  – 9. Summary and concluding discussion  – Appendix. "Bottlenecks for overproduction of proteins in filamentous fungi possibly exist within the secretory pathway, therefore better understanding of this pathway is a key to achieving better yields. “Visible” data with high spatial and temporal resolution of morphology of the hyphal compartments, protein localisation, expression and secretion would need to be added to the existing knowledge to help understand protein secretion and to devise strategies for the improvement of protein production. A series of expression plasmids/cassettes containing a gene encoding the fluorescent protein(s) GFP2 and/or VenusYFP alone or fused to the ER-resident folding chaperone Bip1 and the main cellobiohydrolase I (CBHI) were constructed and introduced into a Trichoderma reesei strain Rut C-30. A transformant strain BV47 expressing the Bip1-Venus fusion protein was applied to visualise the endoplasmic reticulum and potential changes in the ER during Bip1-Venus overexpression. A transformant strain CV48 secreting the main cellobiohydrolase I of T. reesei fused with VenusYFP was used to monitor secretion of the CBHI-Venus fusion protein. In order to investigate the potential interaction between the Bip1 and the secretory protein CBHI, a GFP2/VenusYFP FRET pair system was developed. In the developed FRET system, the transformant strains BG29 expressing Bip1-GFP2, CV48 expressing CBHI-Venus, VG15 expressing Venus-GFP2 and BGCV101 coexpressing Bip1-GFP2 served as the donor, the acceptor, the positive FRET control and the FRET sample, respectively. The ER in the host strain T. reesei Rut C-30 was visualised as a typical network of parallel tubular membranes and some punctate-like bodies through the hyphae. The ER structure in the transformant BV47 expressing the Bip1-Venus fusion protein appeared unusual with an abundance of punctate structures and fewer tubular membranes demonstrating modified spatial organisation of the ER, different to what has been seen in other filamentous fungi studied so far. This type of modification of the ER may assist in forming an ER sub-domain to which overproduced and potentially misfolded proteins can be deposited to wait for further processing. The ER structural modifications appeared to have been caused by…