The effect of induction of protein production was studied in bioreactor cultures of the T. reesei strain Rut-C30 and its transformant expressing endoglucanase I (EGI, Cel7B) fused with a hydrophobic tag. The peptide tag was previously designed for efficient purification of the fusion protein in aqueous two-phase separation. The first phase of the bioreactor cultivations was carried out on glucose containing minimal medium. At the stage when glucose was nearly depleted, the medium was supplemented with rich medium containing lactose as a carbon source to induce production of cellulases. The transformant produced somewhat less secreted protein and cellobiohydrolase I (CBHI, Cel7A) activity than the parental strain. Western analysis of intracellular proteins showed that the fusion protein EGIcore-P5(WP)4 accumulated inside the cell, indicating impaired secretion of the protein. Two-dimensional gel analysis suggested that the fusion protein was possibly trapped early in the secretory pathway. The mRNA levels of the UPR (unfolded protein response) target genes, bip1 and pdil, and the level of the activated hac1 transcript encoding the UPR transcription factor, increased at the same time with an increase in the transcript levels of cellulase genes, suggesting UPR activation in response to cellulase induction. However, only a minor increase in pdi1 and bip1 transcript level was observed in the transformant expressing the fusion protein compared to its parental strain. In addition, slightly lower CBHI production and cbhl mRNA levels were measured in the transformant as compared to the parental strain, indicating activation of the novel repression mechanism of genes encoding secreted proteins in response to secretion stress, RESS (repression under secretion stress).
|Published - 2004
|MoE publication type
|7th European Conference on Fungal Genetics - Copenhagen, Denmark
Duration: 17 Apr 2004 → 20 Apr 2004
|7th European Conference on Fungal Genetics
|17/04/04 → 20/04/04