AEM Accepts, published online ahead of print on 31 August 2007

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Appl. Environ. Microbiol. doi:10.1128/AEM.01196-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Transcriptomics based identification of novel factors enhancing heterologous protein secretion in yeasts

Brigitte Gasser, Michael Sauer, Michael Maurer, Gerhard Stadlmayr, and Diethard Mattanovich^*

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

^* To whom correspondence should be addressed. Email: diethard.mattanovich{at}boku.ac.at.

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 a non-expressing strain. 524 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.

6 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 exoycytosis, proved their benefits for practical application in lab scale production processes by increasing both specific production rates as well as volumetric productivity of an antibody fragment up to 2.5-fold in fed batch fermentations of P. pastoris.

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