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Al-Sheikh, Hashem M.
Languages: English
Types: Unknown
The ability of many filamentous fungi, such as Aspergillus niger, to secret a high level of homologous proteins has led to their consideration as hosts for the production of heterologous proteins. However, the levels of some secreted heterologous proteins have often been low. Although many strategies have been developed to improve the level of secreted heterologous proteins, further studies into the remaining bottlenecks are required. One common strategy used to improve secreted protein production from filamentous fungi is to express the target protein under the control of a highly-induced native promoter. One major bottleneck in the secretion of heterologous proteins is caused by the folding of proteins within the lumen of the endoplasmic reticulum (ER). Recent studies have shown that expressing some heterologous proteins could subject A. niger to ER-stress. In this study, A. niger was subjected to different environmental conditions and ER stress responses were examined under each of these environmental conditions to further investigate the regulation of the gene encoding glucoamylase (glaA). Treating A. niger with dithiothreitol (OTT), a reducing agent that causes the formation of unfolded proteins, caused the down-regulation of transcription of the glaA but not the gene encoding the non-secreted protein y-actin. The OTT-treated fungal cells also showed evidence of induction of the UPR because expression of bipA was up-regulated and splicing of hacA, the gene encoding the transcription factor responsible for induction of the Unfolded Protein Response (UPR), occurs allowing the production of an active HacA protein. This is the first study to show clearly by nuclear run-on studies that the transcriptional down-regulation effect occurs at the level of transcription, rather than mRNA stability, and is found to be mediated through the promoter of the glaA gene (PglaA) in a region more than 1.192 kb upstream of the translational start. As a preliminary attempt to investigate if the transcriptional downregulation effect was mediated through HacA (i.e. part of the UPR), the ER stress was induced through antisense technology to lower the level of POI in the ER of A. niger. Although the transcription of glaA was attenuated in that strain of A. niger, UPR was not evident, suggesting that the transcriptional down-regulation mechanism is controlled differently from the UPR. Furthermore, activation of the ER-Associated Degradation (ERAO) mechanism in OTT-treated A. niger cultures was demonstrated by detecting transcriptional up-regulation of the putative gene encoding the RpnG, a homologue of the yeast Rpn7p subunit of the 26S proteasome.

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