1602 While the economic production of cellulosic ethanol can be tackled by various approaches, protein engineering of cellulases to produce enzymes with improved catalytic activity (Kcat) or enzyme stability is a major issue. In addition, different hosts and environments are screened for novel cellulase activities (Zhang et al. 2006). The engi- neering of enzymes by rational design or directed evolution uses different methodologies to optimize existing enzymes by construction of mutant libraries followed by their testing to identify mutant proteins with improved properties. This involves, e.g., the mutagenesis of potential active center residues and their subsequent kinetic analysis and is based upon the identification of the invariant residues identified in the sequence-based CAZy family classification (http://www. cazy.org; Cantarel et al. 2009). While the methods to generate such mutant libraries are straightforward, there is a bottleneck in the testing and production of the mutant enzymes. Today, such muteins can be routinely expressed in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris, but the functional requirement for modifications such as disulfide bridges or delicate N- and O-glycosylation present at, e.g., the linker domains of the cellulase proteins render most of these expression hosts inappropriate. Exam- ples are the production of the cellobiohydrolase CEL7A in P. pastoris and S. cerevisiae which resulted in hyper- or overglycosylated enzymes with compromised activities (Boer et al. 2000; Godbole et al. 1999; Jeoh et al. 2008; Penttilä et al. 1988; Van Arsdell et al. 1987). The alternative—to produce these mutant enzymes in T. reesei itself—is hampered by the fact that traces of native cellulases are even produced on cellulase-noninducing carbon sources or during sporulation. The resulting protein is therefore contaminated with other enzyme activities which render the kinetic analysis prone to errors (Carle-Urioste et al. 1997). A useful alternative would be the production of mutant cellulases in a host whose cellulase formation has been impaired. Cellulase induction in T. reesei is dependent on the function of the Zn2Cys6 transcriptional regulator XYR1, and knockout mutants in xyr1 are cellulase negative (Stricker et al. 2006). In addition, these cellulases are even not produced during sporulation in such a Δxyr1 strain (Metz et al. 2011). However, it is not yet known whether XYR1 also influences other traits related to cellulase formation such as protein transport or secretion in T. reesei. For example a comparison of the transcriptomic response of an Aspergillus oryzae overproducer of the orthologous xlnR to an xlnR deletion strain did not only reveal changes in the expression pattern of various glycoside hydrolases and genes encoding for pentose catabolism but also changes in various transport processes (Noguchi et al. 2009). In this paper, we therefore tested the hypothesis that a xyr1-knockout strain could be used as a production host for individual cellulase components under constitutive expres- Appl Microbiol Biotechnol (2012) 93:1601–1608 sion signals. Our data show that this strain is a useful vehicle for production of pure cellulases for kinetic analysis. Materials and methods Strains and culture conditions T. reesei QM9414 (ATCC 26921) and the Δxyr1 strain (Stricker et al. 2006) derived from it were used throughout this study. They were maintained on potato dextrose agar (PDA) plates at 28°C. For cellulase expression strains were grown for the indicated time in 250 mL medium in 1 L flasks at 28°C on a rotary shaker at 250 rpm (Multitron 2, Infors AG). A modified medium (Vaheri et al. 1979) containing 10 g/L carbon source, 1.4 g/L (NH4)2SO4, 2.0 g/L KH2PO4, 0.3 g/L MgSO4·7H2O, 0.4 g/L CaCl2·2H2O, 1 g/L peptone, 5 mM urea, 0.02% (w/v) Tween80, and 1/50 (v/v) of the trace element solution (0.25 g/L FeSO4·7H2O, 0.08 g/L MnSO4·H2O, 0.07 g/L ZnSO4·7H2O, 0.1 g/L CoCl2·2H2O) was adjusted to pH 5.0 by citric acid and not further controlled. Expression plasmid construction and fungal transformation Vectors for expression of cel7a (cbh1), cel7b (egl1), and cel12a (egl3) under the tef1 (translation elongation factor 1 alpha) promoter were constructed by inserting the respective PCR amplified cellulase gene in pPtef1-hph (Akel et al. 2009) and named pPtef1-cel7a, -cel7b, or -cel12a. The final expression vector contains 740 bp of the promoter region the T. reesei tef1, followed by the coding and terminator region of the respective cellulase gene and the hygromycin B phosphotransferase (hph) expression cassette as selection m a r k e r. T h e t h r e e c e l l u l a s e g e n e s i n c l u d i n g their respective terminator region were amplified from genomic DNA of T. reesei QM9414 with the primer pairs Egl1_ClaI_F/Egl1_HindIII_R for cel7b, Egl3_ClaI_F/ Egl3_HindIII_R for cel12a Cbh1_ClaI_F and Cbh1_SalI_R for cel7a, thereby introducing the denoted cloning sites (Table 1). For expression under the cdna1 promoter region, the tef1 promoter region was removed by an XhoI/ClaI digest and the PCR amplified, XhoI/ClaI restricted cdna1 promoter region was inserted resulting in pPcdna1-cel12a. For pPcdna1-cel7b construction, the tef1 promoter region was removed from pPtef1-hph by an XhoI/HindIII digest followed by the insertion of the XhoI/ClaI restricted cdna1 promoter region and the ClaI/HindIII restricted cel7b gene via ligation. Fungal transformation was performed as described (Gruber et al. 1990) using uncut plasmid DNA for transformation. Selection media contained 50 μg/mL hygromycin B (Roth). For sporulation, the transformants were transferred to small PDA plates and purified by plating conidiospores onto PDA plates with 0.1% Triton X-100 as colony restrictor. Single