1606 Fig. 3 Cellulase activities in the supernatants of the cel7b, cel7a, and cel12a transformants. Hydrolysis rates of MUC, MULAC, and CMC by culture supernatants of CEL7B (a), CEL12A (b), and CEL7A (c) producing strains respectively were assayed at each sampling point. Volumetric activities (dashed lines) are reflective of total active enzyme amount in the culture supernatant and specific activities (solid lines) are reflective of enzyme purity. Compared to the transformants the parental strain T. reesei Δxyr1 (empty triangle) has negligible activities towards MUC, MULAC, and CMC. The strains selected are identical to Fig. 2 and express the respective cellulase either under the cDNA1 (filled diamond) or tef1 promoter region (filled square). In the case of strain c-cel7b-6, extra D-glucose (10 g/L) was added after 20 h of incubation (empty diamond) Appl Microbiol Biotechnol (2012) 93:1601–1608 Godbole et al. 1999; Nakazawa et al. 2008; Jeoh et al. 2008). The aim of this study was therefore to test whether T. reesei could be used as the native host for overexpression of selected cellulases with high purity and free of other cellulase contaminants. Such cellulase contaminants are inevitable even if the overexpressed protein would be tagged for purification, because cellulases are known to tightly adsorb to each other, particularly in diluted solutions (Sprey and Lamberta 1984). The hypothesis that was tested was whether a strain in which the XYR1 transcriptional regulator of cellulase and hemicellulase gene transcription is nonfunction- al would produce cellulases at a reasonable level and in high purity when fused to a strong expression signal. Using three model cellulase (CEL7A, CEL7B, and CEL12A), we show that strong expression signals such as those from the tef1 and especially the cdna1 expression signals result in a reasonable high production of these enzymes on D-glucose as carbon source. In contrast to earlier reports where the cloning of an expression vector based on the cDNA1 promoter region failed (Nakari-Setälä and Penttilä 1995), we encountered no problem to construct such an expression vector and provide a readily usable expression vector based on the cDNA1 promoter region. We should note that the present studies were performed in shake flasks, and the secreted protein concen- trations reached under these conditions can easily be increased tenfold and more by using controlled fermentation conditions (unpublished data). Interestingly, the Δxyr1 strain was not completely devoid of extracellular protein production and secreted a low amount of extracellular proteins during cultivation on D-glucose. However, our data rule out that these proteins would affect cellulase activity assays under these conditions: all three enzyme activity assays specifically demonstrated activity only in the overexpressing strains and were absent from the Δxyr1 strain. Zymograms for cellulase activity confirmed that the major band detected in the SDS- PAGE nicely overlapped with the cellulase activity (our unpublished results). In addition, the culture filtrates did not reveal any signs of proteolytic degradation which is frequent during cultivation on inducing carbon sources (Haab et al. 1990; Hagspiel et al. 1989). The nature of the two proteins secreted by Δxyr1 has not been examined in this study. However, the lower Mr band exhibits a mass resembling that of the elicitor of plant response protein EPL1. The orthologue of this gene has been shown to be constitutively expressed in another Trichoderma sp., Trichoderma atroviride, during growth on D-glucose (Seidl et al. 2006b), and it is possible that the respective protein produced by Δxyr1 strain is thus T. reesei EPL1. In this study, we used D-glucose as a low-cost carbon source. Due to the use of the Δxyr1 strain as host, carbon sources such as cellulose, xylan, lactose, or D-xylose cannot be applied as substrates since the host strain would fail to assimilate them (Stricker et al. 2006; Seiboth et al. 2007;