ORIGINAL PAPER D. A. MacKenzie Æ T. Guillemette Æ H. Al-Sheikh A. J. Watson Æ D. J. Jeenes Æ P. Wongwathanarat N. S. Dunn-Coleman Æ N. van Peij Æ D. B. Archer UPR-independent dithiothreitol stress-induced genes in Aspergillus niger Received: 19 October 2004 / Accepted: 8 July 2005 / Published online: 14 September 2005 Ó Springer-Verlag 2005 Abstract A subtraction library was prepared from cul- tures of Aspergillus niger that had or had not been ex- posed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn 2+ -containing superoxide dismu- tase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control con- ditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stres- ses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined. Keywords PCR subtraction Æ Reducing agent Æ Protein secretion Æ Methyltransferases Æ Filamentous fungi Introduction Aspergillus niger is used as a host for production and secretion of many native and heterologous proteins on an industrial scale. Despite its efficient protein secretion system for endogenous proteins, yields of heterologous proteins are generally poorer, and this has engendered interest in improving yields by genetically manipulating the secretory pathway (Conesa et al. 2001; MacKenzie et al. 2004). The isolation and study of genes, particu- larly those thought to represent blocks to the secretion of heterologous proteins, will be required for a full understanding of the secretion pathway. Several secre- tion-related genes have been manipulated in A. niger and their effects on protein yields characterised. These in- clude the genes encoding protein disulphide isomerase, pdiA (Ngiam et al. 2000; Moralejo et al. 2001; Al-Sheikh et al. 2004), immunoglobulin-binding protein, bipA (Conesa et al. 2002; Lombran˜a et al. 2004), cyclophilin B, cypB (Wiebe et al. 2001) and calnexin, clxA (Conesa et al. 2002; Wang et al. 2003). With a view to isolating further genes of importance to the secretion pathway, a modified, PCR-augmented, Communicated by E. Cerda´-Olmedo D. A. MacKenzie (&) Æ D. J. Jeenes Department of Food Safety Science, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK E-mail: donald.mackenzie@bbsrc.ac.uk Tel.: +44-1603-255255 Fax: +44-1603-255288 T. Guillemette Æ H. Al-Sheikh Æ A. J. Watson Æ D. B. Archer School of Biology, University of Nottingham, University Park, Nottingham NG7 2RD, UK P. Wongwathanarat Department of Biotechnology, Faculty of Science and Technology, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12121, Thailand N. S. Dunn-Coleman Genencor International Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA N. van Peij Research and Development, DSM Food Specialties, PO Box 1, 2600 Delft, The Netherlands Present address: A. J. Watson School of Biology, University of Newcastle-upon-Tyne, King George VI Building, Newcastle-upon-Tyne NE1 7RU, UK Mol Gen Genomics (2005) 274: 410–418 DOI 10.1007/s00438-005-0034-3