Unfolded Protein Response in Aspergillus niger chemostat fermentations.

T. Goosen 1,2, T. R. Jørgensen 3, J.J.L. Iversen 3, C.A.M.J.J. van den Hondel1,2.

Author address: 

1Leiden University, Institute of Biology Leiden, Fungal Genetics Research Group, W assenaarseweg 64, 2333 AL Leiden, The Netherlands, 2Department of Microbiology, TNO-Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, The Netherlands, 3Depar

Abstract: 

Unfolded Protein Response (UPR) is a universal reaction of eukaryotic cells to protein folding stress in the endoplasmic reticulum (ER). The expression of heterologous secreted proteins often results in such stress and thus UPR, also in filamentous fungi like Aspergillus niger which are renowned for their high secretion capacity. As a consequence, the production yield of secreted heterologous proteins usually is very low (milligrams/L at best). UPR results in induction of expression of genes that allow the cell to cope with the surplus of protein folding intermediates. Identification of these genes and understanding the response pathway(s) should give leads to improve the folding and secretion capacity of A. niger. Towards this, two approaches are taken: transcriptomics with Affymetrix genome arrays to identify up- or down-regulated genes and genetic screening to select regulatory mutants. For transcriptomics analysis, A. niger strains are constructed in which poorly folded (scFv) or unfoldable (yeast CPY*) proteins can be expressed under control of the regulated glaA promoter. RNA analysis is first performed with shake flask cultures, both under induced and non-induced conditions for the expression of the poorly folded proteins For a sound transcriptomics analysis, tight control of all cultivation conditions is an absolute prerequisite. W e therefore are developing protocols for steady-state fermentation of A. niger under repressing conditions for the glaA promoter and for transition to a glaA induced steady-state. The samples collected throughout these fermentations are used for transcriptomic and protein analysis. Tools are developed to improve the genetic screen for the isolation of regulatory mutants with altered UPR characteristics. W e will report on the recent progress made in these project goals. This research is carried out within the Kluyver Centre for Genomics of Industrial Fermentation.
2005

abstract No: 

155.

Full conference title: 

23rd Fungal Genetics Conference
    • Fungal Genetics Conference 23rd (2002)