Expression and Secretion of Barley Cysteine Endopeptidase B and Cellobiohydrolase I in Trichoderma reesei

Abstract

Localization of expression and secretion of a heterologous barley cysteine endopeptidase (EPB) and the homologous main cellobiohydrolase I (CBHI) in a Trichoderma reesei transformant expressing both proteins were studied. The transformant was grown on solid medium with Avicel cellulose and lactose to induce the cbh1 promoter for the synthesis of the native CBHI and the recombinant barley protein linked to a cbh1 expression cassette. Differences in localization of expression between the two proteins were clearly indicated by in situ hybridization, indirect immunofluorescence, and immunoelectron microscopy. In young hyphae, native-size recombinant epb mRNA was localized to apical compartments. In older cultures, it was also seen in subapical compartments but not in hyphae from the colony center. The recombinant EPB had a higher molecular weight than the native barley protein, probably due to glycosylation and differential processing in the fungal host. As was found with its transcripts, recombinant EPB was localized in apical and subapical compartments of hyphae. The cbh1 mRNA and CBHI were both localized to all hyphae of a colony, which suggests that the endogenous CBHI was also secreted from these. In immunoelectron microscopy, the endoplasmic reticulum and spherical vesicles assumed to contribute to secretion were labeled by both CBHI and EPB antibodies while only CBHI was localized in elongated vesicles close to the plasma membrane and in hyphal walls. The results indicate that in addition to young apical cells, more mature hyphae in a colony may secrete proteins.

Codon optimization of xylanase genexynBfrom the thermophilic bacteriumDictyoglomus thermophilumfor expression in the filamentous fungusTrichoderma reesei

“…Selected T. reesei transformants were plated on minimal medium containing Avicel cellulose and lactose [18] and incubated at 28°C for 7 days to induce the cbh1 promoter. Incubation was then continued at 70°C overnight to inactivate the endogenous hydrolases produced by Trichoderma and xylanase activity detected as described for E. coli recombinants.…”

“…Two T. reesei transformants exhibiting a large clearing halo in the plate test, indicating production of thermostable xylanase activity, were chosen for Northern analysis. Induction cultures were grown on Avicel lactose plates [18] incubated at 28°C for 7 days and mRNA was isolated using the FastRNA™ kit (Bio 101, USA) following the manufacturer's instructions. Northern blotting [12] was carried out using the DIG‐labeled (Roche, Germany) 0.6‐kb xynB fragment as a probe, as recommended by the manufacturer.…”

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei

“…All plates were incubated for 5^10 days for the transformants to emerge. Selected T. reesei transformants were plated on minimal medium containing Avicel cellulose and lactose [18] and incubated at 28³C for 7 days to induce the cbh1 promoter. Incubation was then continued at 70³C overnight to inactivate the endogenous hydrolases produced by Trichoderma and xylanase activity detected as described for E. coli recombinants.…”

“…Two T. reesei transformants exhibiting a large clearing halo in the plate test, indicating production of thermostable xylanase activity, were chosen for Northern analysis. Induction cultures were grown on Avicel lactose plates [18] incubated at 28³C for 7 days and mRNA was isolated using the FastRNA1 kit (Bio 101, USA) following the manufacturer's instructions. Northern blotting [12] was carried out using the DIG-labeled (Roche, Germany) 0.6-kb xynB fragment as a probe, as recommended by the manufacturer.…”

Expression of Barley Endopeptidase B in Trichoderma reesei

“…Interestingly, an enzyme that has not yet been identified which is capable of correct processing of CBHI-Fab fusion protein in T. reesei Rut-C30-based transformants has been reported by Nyyssönen et al (17). Evidence exists that the barley EPB is glycosylated in T. reesei, which may interfere with the processing of the recombinant enzyme into an active form (16).…”