We have taken a holistic approach into analysis of heterologous gene expression in Trichoderma involving enzyme activity measurements, gene transcription assays, zymogram gels, Western blotting and proteomics. The T. reesei transformant expressing the bacterial xynB gene encoding a thermophile xylanase was grown in a laboratory fermenter and assays were performed on culture supernatants collected from different time points. Transcription of the xynB under the T. reesei cbhl (cellobiohydrolase 1) promoter was comparable to that of the native cbh1, however, the yield of the heterologous xylanase produced in this particular transformant was no more than about 100 mg/l. Transcription analysis of selected genes involved in UPR (unfolded protein response) indicated a slight increase in the expression of pdi (protein disulfide isomerase) but otherwise the UPR pathway seemed not have been induced. Comparative proteomic analysis of the culture supernatant from the non-transformed host and the transformant expressing thermophilic xylanase showed differential expression of 23 proteins. Analysis of these spots is underway. This multifaceted approach may reveal crucial proteins involved in heterologous gene expression in filamentous fungi in general and Trichoderma in particular.
|Title of host publication||Poster Abstracts|
|Publication status||Published - 2004|
|Event||7th European Conference on Fungal Genetics - Copenhagen, Denmark|
Duration: 17 Apr 2004 → 20 Apr 2004
|Conference||7th European Conference on Fungal Genetics|
|Period||17/04/04 → 20/04/04|
Nevalainen, H., Hekelaar, J., Uusitalo, J., Te o, J., Jonkers, I., Bergquist, P., & Penttilä, M. (2004). Transcriptional and proteomic changes in Trichoderma reesei expressing a heterologous bacterial xynB gene from the thermophile Dictyoglomus thermophilum. In Poster Abstracts (pp. 201). [VIIIp-30] http://www.fgsc.net/ecfg7/PostersVIIIFungalCellFactories.pdf