| Summary: | Secreted amounts of endogenous enzymes, such as cellobiohydrolase I (CBHI) produced by Trichodenna reesei, are superior to any organism studied so far. CBHI, the heterologous barley cysteine proteinase (EPB), calf chymosin and a fusion protein consisting of chymosin joined to the CBHI core-linker region were produced under the cbh1 promoter in a T. reesei mutant Rut-C30. The aim was to reveal secretory bottlenecks for the heterologous proteins. At the mRNA level, the expression of epb and cbh1 differed clearly. Transcripts were localised synchronously by in situ hybridisation to the same compartments as their respective proteins, except that the mRNAs were absent from the hyphal tips. The 42 kDa heterologous EPB pro-protein was N-glycosylated and sequentially processed by a postulated Golgi-resident Kex2p-like peptidase to a secreted polypeptide of 38.5 kDa, and thereafter to polypeptides of 37.5, 35.5 and 32 kDa exhibiting proteinase activity. The final 32 kDa EPB was four times less active than the unglycosylated 30 kDa barley EPB. This was shown to result from N-glycosylation which led to incomplete EPB maturation in Trichoderma. All the examined heterologous proteins and CBHI were shown to be secreted into the culture medium and were visualised by immuno microscopy in the ER, Golgi, secretory vesicles, plasma membrane and also in cell wall but not in septae. CBHI, chymosin and the chymosin fusion protein occurred throughout the hyphal length in contrast to EPB which was detected only at growing apical cells. Immuno-EM morphometry of chymosin and CBHI signals in the cytoplasm and cell wall was applied to correlate the signals to volumes of the mature hyphal cell. The total amount of signals in the cell wall were 4 %, 83 % and 49 % for CBHI, chymosin and for the chymosin fusion protein, respectively. This result indicates that fusion of the chymosin to an endogenous carrier prevented the adhesion of chymosin in the cell wall.
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