Appl Microbiol Biotechnol (2012) 93:1601–1608 DOI 10.1007/s00253-011-3674-8 APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background Fatma Uzbas & Ugur Sezerman & Lukas Hartl & Christian P. Kubicek & Bernhard Seiboth Received: 18 August 2011 / Revised: 6 October 2011 / Accepted: 26 October 2011 / Published online: 13 November 2011 # The Author(s) 2011. This article is published with open access at Springerlink.com Abstract Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on D- glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background F. Uzbas : U. Sezerman Biological Sciences and Bioengineering, Sabanci University, Orhanli, Tuzla, Istanbul, Turkey L. Hartl : C. P. Kubicek : B. Seiboth (*) Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, 166-5, 1060 Vienna, Austria e-mail: bseiboth@mail.tuwien.ac.at essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background. Keywords Cellulase . Recombinant protein production . Hypocrea jecorina . xyr1 . cDNA1 . tef1 Introduction Lignocellulose, composed mainly of cellulose, hemicellulose, and lignin, is the most abundant renewable carbon source on earth and therefore an attractive resource to use it for the production of different chemical building blocks or biofuels. Due to the complex structure and the recalcitrance of lignocellulosic feedstocks, the conversion to their monomers is much more difficult to achieve than using, e.g., starch-based feedstocks (Himmel et al. 2007). As a result, the breakdown of the polymeric sugars, especially cellulose, requires large amounts of enzymes (Merino and Cherry 2007). Recent demands to reduce the production costs for biofuels from lignocellulosic plant matter led to a general renaissance in cellulase research (Carroll and Somerville 2009; Wilson 2009; Kumar et al. 2008). Fungal cellulases are attractive for enzymatic cellulose conversion as they are highly active and can be expressed at levels exceeding 100 g per liter in fungal hosts such as Trichoderma reesei (Hypocrea jecorina, Cherry and Fidantsef 2003; Kubicek et al. 2009). To date, many important commercial enzyme preparations for biomass conversion of cellulose or hemicelluloses are from T. reesei. These T. reesei cellulase mixes include different types of cellulases including cellobiohydrolases (CEL7A and CEL6A), endoglucanases (e.g., CEL7B and CEL6B) and different β-glucosidases (e.g., CEL3A) which synergistically degrade cellulose (Henrissat et al. 1985).