Protein production in filamentous fungi: stress responses and improvement by genetic engineering

Industrially exploited filamentous fungi are known for their extremely high capacity of protein secretion. High loads of protein, and especially foreign protein, in the secretory pathway form a challenge to the production organism and expose it to secretion stress. Unfolded protein response (UPR) denotes the induction mechanism of genes encoding ER-resident chaperones and foldases and numerous other genes involved in protein secretion. This induction is triggered when unfolded proteins accumulate into the ER. We have investigated several components of the UPR pathway, its regulatory range and its utilisation for improvement of protein production in filamentous fungi, predominantly Trichoderma reesei. We have shown that the UPR transcription factor gene hal1 is activated in filamentous fungi by a dual mechanism operational at the mRNA level. This mechanism includes a splicing event of an unconventional intron of only 20 nt in length and a truncation of the mRNA at the 5' flanking region. This truncation removes an upstream open reading frame from the mRNA, and we have shown that these uORFs are involved in translational control of the HAC1 protein formation. The regulatory range of secretion stress responses in T. reesei has been studied by subtraction library cloning, cDNA-AFLP and proteomics. These methods have revealed a high number of genes up-regulated by secretion stress, including ones involved in protein folding, glycosylation and trafficking in the secretory pathway. An interesting link between amino acid metabolism and secretion stress was also observed. The activated form of the hac1/hacA transcription factor gene was expressed from a constitutive promoter in T. reesei, Aspergillus niger var. awamori and S. cerevisiae, and in all cases the induction of the UPR pathway was observed. In A. niger the production of Trametes versicolor laccase was improved about 5-fold and that of bovine chymosin about 3-fold by the constitutive UPR induction. The production of Bacillus alpha-amylase and native invertase was enhanced in S. cerevisiae with this strategy. We have observed that concurrently with the induction of the UPR pathway, the genes encoding secreted proteins are rapidly down-regulated in T. reesei. This type of regulation can be caused by different secretion inhibitors and by foreign protein expression. The down-regulation is dependent on the promoter of the affected gene, suggesting that it is functional at the transcriptional level.