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Applied and Environmental Microbiology, February 2000, p. 775-782, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Characterization of a Foldase, Protein Disulfide Isomerase A, in the Protein Secretory Pathway of Aspergillus niger

Celina Ngiam,1,dagger David J. Jeenes,1,* Peter J. Punt,2 Cees A. M. J. J. Van Den Hondel,2 and David B. Archer1

Division of Food Safety Sciences, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom,1 and TNO Nutrition and Food Research Institute, Department of Molecular Genetics and Gene Technology, 3700 AJ Zeist, The Netherlands2

Received 31 August 1999/Accepted 3 November 1999

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Delta pdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

* Corresponding author. Mailing address: Division of Food Safety Sciences, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Phone: 44-1603-255255. Fax: 44-1603-507723. E-mail: david.jeenes{at}bbsrc.ac.uk.

dagger Present address: Gene Therapy Laboratory, Norris Cancer Center, University of Southern California School of Medicine, Los Angeles, Calif.

Applied and Environmental Microbiology, February 2000, p. 775-782, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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