1604 Results centrifuged at 5,000×g for 10 min, the pellet was resuspended with ddH2O to get rid of salts and centrifuged again. The pellets were dried in an oven (70°C) to constant weight for 1 week. Cellulase expression vector construction For the expression of cellulases in a Δxyr1 strain, we had to switch their endogenous promoter regions. We chose the promoters of two genes that were isolated during the screening for highly expressed genes during growth on D- glucose as carbon source—i.e., tef1 (translation elongation factor 1-α-encoding; abbreviated as “t”) and the uncharac- terized “cdna1” (abbreviated as “c,” Nakari et al. 1993; Nakari-Setälä and Penttilä 1995). With the help of the previously released T. reesei genome sequence, it was now possible to localize the complete cDNA1 gene on scaffold 23:43726–44652 (http://genome.jgi-psf.org/Trire2/Trire2. home.html). In agreement with earlier results, a high number of ESTs are assigned to the cDNA1 gene. BLASTP search of the translated nucleotide sequence showed that the protein is only moderately conserved showing a 39 to maximal 46% aa identity to other fungal hypothetical proteins from Nectria haematococca, Gibberella zeae, Verticillium alboatrum, Magnaporthe grisea, Sclerotinia sclerotiorum, and Botryotinia fuckeliana. Three cellulase genes were chosen for testing cellulase expression in the Δxyr1 background in T. reesei: one cellobiohydrolase encoded by cel7a (cbh1) and two endoglucanases encoded by cel7b (egl1) and cel12a (egl3). cel7b and cel12a were expressed under both promoters, whereas cel7a was tested only with the tef1 promoter region. All three cellulases together with their terminator region were amplified by PCR and ligated downstream of the respective promoter region (Fig. 1) Recombinant expression of three cellulases in the Δxyr1 strain After transformation of the Δxyr1 strain with different expression plasmids and purification of putative trans- formants, the integration of the expression vector was tested by PCR and Southern analysis (data not shown). Strains with one additional copy of the expression cassette were chosen for further analysis and named according to the promoter region and cellulase expressed (e.g., t-cel7b for a transformant expressing cel7b under the tef1 promoter region). Their growth rate on glucose was similar to the parental strain. Strains produced 2.5 g/L dry biomass after Appl Microbiol Biotechnol (2012) 93:1601–1608 Fig. 1 Expression plasmids for cellulase production. Schematic presen- tation of the two cellulase expression plasmids used in this study. Both plasmids contain the hygromycin B expression cassette as fungal selection marker followed by either the tef1 or the cDNA1 promoter region and the coding and terminator region of the respective cellulase gene (e.g., the endoglucanase encoding gene cel7b) 20 h and reached a maximum of 5 g/L after 30 h of cultivation. To test for cellulase production in these strains, we followed the total extracellular protein production over time: all strains secreted protein into the medium until around 36 h of cultivation. Best transformants reached extracellular protein concentrations of more than 40 mg/L, which exceeds the Δxyr1 recipient strain which formed up to 18 mg/L protein as background (Fig. 2). To visualize cellulase expression, the proteins in the culture filtrate were separated by SDS-PAGE: Consistent with previous findings (e.g., Jeoh et al. 2008; Shoemaker et al. 1983; Sprey and Uelker 1992), CEL7B is detected as a broad band between 60 and 70 kDa (Fig. 2a), CEL12A is found around 25 kDa (Fig. 2b), and CEL7A, which is found around 65 kDa, was evident from a band around 70 kDa in the gels (Fig. 2c). No degradation products of the recombinant produced cellu- lases which would have been evident from a decrease of the cellulase band or an increase in bands with lower molecular mass with time were observed. In the Δxyr1 control background, only two faint protein bands appeared after 28 h of cultivation which were only visible when a higher volume of culture supernatant was used (Fig. 2a, c). Quantification of cellulase expression in the Δxyr1 strain To test whether the secreted cellulases were active, CEL7A and CEL7B enzymes in the culture filtrates were quantified using the specific fluorogenic substrates MULAC (van Tilbeurgh et al. 1982) or MUC (Claeyssens and Aerts 1992), respectively. The results, shown in Fig. 3a, indicate that only the CEL7B producing transformants show activity towards MUC, strain c-cel7b-6 thereby giving highest