This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gasser, B.
Right arrow Articles by Mattanovich, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gasser, B.
Right arrow Articles by Mattanovich, D.
Agricola
Right arrow Articles by Gasser, B.
Right arrow Articles by Mattanovich, D.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, October 2007, p. 6499-6507, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptomics-Based Identification of Novel Factors Enhancing Heterologous Protein Secretion in Yeasts{triangledown}

Brigitte Gasser,1 Michael Sauer,1,2 Michael Maurer,1,2 Gerhard Stadlmayr,1,2 and Diethard Mattanovich1,2*

Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria,1 School of Bioengineering, University of Applied Sciences FH—Campus Vienna, Vienna, Austria2

Received 29 May 2007/ Accepted 18 August 2007

Efficient production of heterologous proteins with yeasts and other eukaryotic hosts is often hampered by inefficient secretion of the product. Limitation of protein secretion has been attributed to a low folding rate, and a rational solution is the overexpression of proteins supporting folding, like protein disulfide isomerase (Pdi), or the unfolded protein response transcription factor Hac1. Assuming that other protein factors which are not directly involved in protein folding may also support secretion of heterologous proteins, we set out to analyze the differential transcriptome of a Pichia pastoris strain overexpressing human trypsinogen versus that of a nonexpressing strain. Five hundred twenty-four genes were identified to be significantly regulated. Excluding those genes with totally divergent functions (like, e.g., core metabolism), we reduced this number to 13 genes which were upregulated in the expression strain having potential function in the secretion machinery and in stress regulation. The respective Saccharomyces cerevisiae homologs of these genes, including the previously characterized secretion helpers PDI1, ERO1, SSO2, KAR2/BiP, and HAC1 as positive controls, were cloned and overexpressed in a P. pastoris strain expressing a human antibody Fab fragment. All genes except one showed a positive effect on Fab fragment secretion, as did the controls. Six out of these novel secretion helper factors, more precisely Bfr2 and Bmh2 (involved in protein transport), the chaperones Ssa4 and Sse1, the vacuolar ATPase subunit Cup5, and Kin2 (a protein kinase connected to exocytosis), proved their benefits for practical application in laboratory-scale production processes by increasing both specific production rates and the volumetric productivity of an antibody fragment up to 2.5-fold in fed-batch fermentations of P. pastoris.

* Corresponding author. Mailing address: Institute of Applied Microbiology, Department of Biotechnology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, A-1190 Vienna, Austria. Phone: 43-1-36006-6569. Fax: 43-1-3697615. E-mail: diethard.mattanovich{at}boku.ac.at

{triangledown} Published ahead of print on 31 August 2007.

Applied and Environmental Microbiology, October 2007, p. 6499-6507, Vol. 73, No. 20
0099-2240/07/$08.00+0     doi:10.1128/AEM.01196-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Raimondi, S., Zanni, E., Talora, C., Rossi, M., Palleschi, C., Uccelletti, D. (2008). SOD1, a New Kluyveromyces lactis Helper Gene for Heterologous Protein Secretion. Appl. Environ. Microbiol. 74: 7130-7137 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»