Stress effects caused by the expression of a mutant cellobiohydrolase I and proteasome inhibition in Trichoderma reesei Rut-C30

• Published in: New biotechnology 2013-01, Vol.30 (2), p.183-191
• Main Authors: Kautto, Liisa, Grinyer, Jasmine, Paulsen, Ian, Tetu, Sasha, Pillai, Aneesh, Pardiwalla, Swapneel, Sezerman, Ugur, Akcapinar, Gunseli Bayaram, Bergquist, Peter, Te’o, Junior, Nevalainen, Helena
• Format: Article
• Language: English
• Subjects: Cellobiohydrolase I; cellulose 1,4-beta-cellobiosidase; Cellulose 1,4-beta-Cellobiosidase - genetics; Cellulose 1,4-beta-Cellobiosidase - metabolism; confocal laser scanning microscopy; DNA; Endoplasmic Reticulum-Associated Degradation - drug effects; Endoplasmic Reticulum-Associated Degradation - genetics; fluorescence microscopy; Fluorescent Antibody Technique; Fungal Proteins - secretion; fungi; gene expression regulation; Gene Expression Regulation, Fungal - drug effects; genes; Genes, Fungal - genetics; hyphae; Hyphae - drug effects; Hyphae - enzymology; Hypocrea jecorina; Leupeptins - pharmacology; Mutant Proteins - metabolism; mutants; mutation; Mutation - genetics; proteasome endopeptidase complex; Proteasome Endopeptidase Complex - metabolism; Proteasome Inhibitors - pharmacology; protein secretion; proteins; quality control; quantitative polymerase chain reaction; reverse transcriptase polymerase chain reaction; Stress, Physiological - drug effects; Transcription, Genetic - drug effects; transcriptomics; Trichoderma - drug effects; Trichoderma - enzymology; Trichoderma - genetics; Trichoderma - physiology; Trichoderma reesei; Venus; yields
• ISSN: 1871-6784; 1876-4347
• DOI: 10.1016/j.nbt.2012.07.005

► A massive upregulation of UPR and ERAD-related genes was observed upon overexpression misfolded CBHI. ► Overexpression of mutant CBHI caused physiological stress in the fungal hyphae. ► The mutant CBHI accumulated in the ER and colocalized with the proteasome. ► Inhibition of the proteasome with MG132 was specific to the proteasome-associated genes. 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 CBHIΔ4-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.