New Biotechnology  Volume 30, Number 2  January 2013 RESEARCH PAPER Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30 Liisa Kautto 1,2, , Jasmine Grinyer 1,2 , Ian Paulsen 1,2 , Sasha Tetu 1,2 , Aneesh Pillai 1 , Swapneel Pardiwalla 1 , Ugur Sezerman 3 , Gunseli Bayaram Akcapinar 3 , Peter Bergquist 1,2,4 , Junior Te’o 1,2 and Helena Nevalainen 1,2 1 Department of Chemistry and Biomolecular Sciences, Faculty of Science, Macquarie University, Macquarie University, NSW 2109, Australia 2 Biomolecular Frontiers Research Centre, Macquarie University, NSW 2109, Australia 3 Sabanci University, Biological Sciences and Bioengineering Prog., 34956 Orhanli Tuzla, Istanbul, Turkey 4 Department of Molecular Medicine & Pathology, University of Auckland Medical School, Auckland, New Zealand Trichoderma reesei Rut-C30 is used widely as an expression host for various gene products. We have explored cellular effects caused by the expression of a mutant form of cellobiohydrolase I (CBHI), the major secreted protein of T. reesei using biochemical and transcriptomic analyses and confocal laser scanning microscopy. The mutated CBHI was tagged fluorescently with Venus to establish the subcellular location of the fusion protein and its potential association with the proteasome, an organelle assigned for the disposal of misfolded proteins. Expression of the mutant CBHI in the high protein- secreting host Rut-C30 caused physiological changes in the fungal hyphae, affected protein secretion and elicited ER stress. A massive upregulation of UPR- and ERAD-related genes sec61, der1, uba1, bip1, pdi1, prp1, cxl1 and lhs1 was observed by qRT-PCR in the CBHID4-Venus strain with four mutations introduced in the DNA encoding the core domain of CBHI. Further stress was applied to this strain by inhibiting function of the proteasome with MG132 (N-benzoylcarbonyl(Cbz)-Leu-Leu-leucinal). The effect of MG132 was found to be specific to the proteasome-associated genes. There are no earlier reports on the effect of proteasome inhibition on protein quality control in filamentous fungi. Confocal fluorescence microscopy studies suggested that the mutant CBHI accumulated in the ER and colocalized with the fungal proteasome. These results provide an indication that there is a limit to how far T. reesei Rut-C30, already under secretion stress, can be pressed to produce higher protein yields. Introduction A major limitation to produce high amounts of foreign proteins in filamentous fungi including Trichoderma reesei is the accumulation of misfolded proteins that cause considerable stress for the host cells (reviewed in [1]). Understanding the physiological effects and molecular biology of conformational stress (ER stress) is thus a priority when aiming at effective use of high protein-secreting fungal strains for the production of recombinant proteins of industrial and medical interest. A commonly used expression host is T. reesei Rut-C30 (reviewed in [2–5]), which was obtained as a result of three rounds of random mutagenesis of the wild type QM6a and screening for cellulase overproduction and catabolite repression [6]. Sequencing of the Rut-C30 genome [5,7] has revealed several changes compared to the wild type such as the loss of over 100 kb of genomic DNA. The missing DNA fragments house about 33 genes among which are genes that encode proteins involved in vesicle trafficking and vacuolar sorting. There are also physiological changes including a higher content of endoplasmic reticulum [8], apparent lack of typical Golgi bodies [9], and indications of impaired osmotic Research Paper Corresponding author: Kautto, L. (liisa.kautto@mq.edu.au) 1871-6784/$ - see front matter ß 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.nbt.2012.07.005 www.elsevier.com/locate/nbt 183