Applied genetics and molecular biotechnology

Applied Microbiology and Biotechnology

, Volume 93, Issue 4, pp 1601-1608

First online:

Open Access This content is freely available online to anyone, anywhere at any time.

A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

  • Fatma UzbasAffiliated withBiological Sciences and Bioengineering, Sabanci University
  • , Ugur SezermanAffiliated withBiological Sciences and Bioengineering, Sabanci University
  • , Lukas HartlAffiliated withResearch Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology
  • , Christian P. KubicekAffiliated withResearch Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology
  • , Bernhard SeibothAffiliated withResearch Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering, Vienna University of Technology Email author 

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 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