| Popis: |
Understanding and optimizing the protein secretion in the industrial workhorse Trichoderma reesei is of major interest. In the filamentous fungus, the secretion of heterologous proteins or high production rates of native enzymes in the strain RUT-C30 were observed to induce the ‘unfolded protein response’ (UPR). The UPR is triggered by an accumulation of un- or misfolded proteins in the endoplasmic reticulum (ER) when the protein processing capacity of the ER is surpassed. To reduce the ER stress, the UPR up-regulates the secretion machinery, but the expression of several secreted enzymes is repressed as well. More research is required regarding this so-called ‘repression under secretion stress’ (RESS) and the UPR in T. reesei. Here, the UPR and RESS were investigated on the transcriptional level by reverse transcription quantitative PCR (RT-qPCR). Transcript levels of genes encoding (hemi-)cellulases, UPR indicators and (hemi-)cellulase regulators were monitored in a wild-type strain and two Xyr1 overexpression strains. One overexpression strain produces the wild-type Xyr1 and the other a modified Xyr1 version. The enhanced enzyme secretion in these strains was speculated to induce the UPR. However, the present thesis suggests that the elevated enzyme secretion does not activate the UPR. This was indicated by similarly low mRNA levels of the UPR indicators (hac1, bip1) in the wild-type strain and the Xyr1 overexpression strains. To reveal the mediators and regulatory mechanisms of RESS, we investigated the involvement of the (hemi-)cellulase activators Xyr1, Rxe1 and Ace3 in an indirect induction of RESS. To this end, ER stress was induced by the addition of dithiothreitol (DTT). The transcription factor Ace3 seems to be neither a RESS-target nor -mediator, as no significant impact of ER stress on ace3 transcription was observed. The involvement of Rxe1 in RESS as a target or mediator remains unclear due to inconsistent rxe1 transcript levels in the three strains investigated. Xyr1 appears also not to be indirectly mediating RESS: the transcription of (hemi-)cellulase encoding genes was down-regulated during UPR even when the (hemi-)cellulase activator Xyr1 was overexpressed. Whether the xyr1 gene is a UPR- or RESS-target might depend on the carbon source, as a clear decline of xyr1 mRNA levels under ER stress was solely observed on lactose. Different mechanisms for the down-regulation of secreted enzymes depending on the cultivation condition might exist in T. reesei. On the one hand, transcript levels of hemicellulose- and cellulose-degrading enzymes showed a different temporal decrease during ER stress. On the other hand, the absolute change of transcript levels of the genes encoding UPR indicators and transcriptions factors was different for xylan- and lactose-grown samples. In addition, this thesis suggest a mutual regulatory influence between Xyr1 and Rxe1 (regulator 1 of Xyr1 expression), because transcript levels of rxe1 were enhanced by factor of 10 by the overexpression of wild-type Xyr1. |
