Abstract
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.
Original language | English |
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Publication status | Published - 2004 |
Event | Workshop on Production and Characterization of Foreign Proteins in Fungal Hosts - Ieper, Belgium Duration: 25 Aug 2004 → 27 Aug 2004 |
Workshop
Workshop | Workshop on Production and Characterization of Foreign Proteins in Fungal Hosts |
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Country/Territory | Belgium |
City | Ieper |
Period | 25/08/04 → 27/08/04 |