Protein production in filamentous fungi: cellular responses and strain improvement

In recent years our understanding of cellular responses in Trichoderma and Aspergillus towards secretion load has increased significantly. Production of large amounts of native proteins or production of difficult foreign proteins provokes secretion stress that is believed in particular to be caused by folding limitations within the ER. Impairnment of folding causes the so called unfolded protein response (UPR), which is mediated by the regulatory protein HAC that causes modification of expression of a number of genes involved in various cellular processes related to protein secretion. In addition to the classical UPR, also other cell responses have been discovered using genome-wide approaches. Furthermore, a novel mechanism (RESS) has been identified that down-regulates expression of genes encoding native secreted proteins such as cellulases and glycoamylase under secretion stress conditions. These results show that the function of the secretory pathway is interlinked with other cellular processes, and that several feed-back mechanisms may exist that serve to balance between expression, folding, glycosylation and secretion of proteins. It is possible to alter these cellular mechanisms and create improved production strains. For instance, overexpression of HACA increases production of calf chymosin and laccase in A.niger. The EC-funded Eurofung consortium has studied protein secretion in filamentous fungi and some of the results obtained will also be presented. References: Pakula, T.M., Laxell, M., Huuskonen, A., Uusitalo, J., Saloheimo, M. & Penttilä, M. 2003. The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells. J. Biol. Chem. 278, 45011-45020. Saloheimo, M., Valkonen, M. & Penttilä, M. 2003. Activation mechanisms of the HACI-mediated unfolded protein response in filamentous fungi. Mol. Microbiol. 47, 1149 1161. Valkonen, M., Penttilä, M. & Saloheimo, M. 2003. Effects of inactivation and constitutive expression of the unfolded-protein response pathway on protein production in the yeast Saccharomyces cerevisiae. Appl. Env. Microb. 69, 2065-2072. Valkonen, M., Ward, M., Wang, H., Penttilä, M. & Saloheimo, M. 2003. Improvement of foreign-protein production in Aspergillus niger var. awamori by constitutive induction of the unfolded-protein response. Appl. Env. Microb. 69, 6979-6986. Mulder, H. J., Saloheimo, M., Penttilä, M. & Madrid, S.M. The transcription factor HACA mediates the unfolded protein response in Aspergillus niger, and upregulates the transcription of its own gene. Mol. Gen. Genet., in press.