Nucleic Acids Research Advance Access published online on June 6, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn369
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Methods Online |
Promoter library designed for fine-tuned gene expression in Pichia pastoris
1Institute of Molecular Biotechnology, Graz University of Technology, 2Research Centre Applied Biocatalysis, Petersgasse 14/2, 8010 Graz, Austria, 3Institute of Biotechnology, Zurich University of Applied Sciences (ZHAW), Grüental, 8820 Wädenswil, Switzerland, 4Department of Biological Sciences, University of the Pacific, Stockton, CA 95211 and 5Keck Graduate Institute of Applied Life Sciences, 535 Watson Drive, Claremont, CA 91711, USA
*To whom correspondence should be addressed. Tel: +43 316 873 4074; Fax: +43 316 873 4071; Email: a.glieder{at}tugraz.at
Received March 19, 2008. Revised May 6, 2008. Accepted May 23, 2008.
Although frequently used as protein production host, there is only a limited set of promoters available to drive the expression of recombinant proteins in Pichia pastoris. Fine-tuning of gene expression is often needed to maximize product yield and quality. However, for efficient knowledge-based engineering, a better understanding of promoter function is indispensable. Consequently, we created a promoter library by deletion and duplication of putative transcription factor-binding sites within the AOX1 promoter (PAOX1) sequence. This first library initially spanned an activity range between 
6% and >160% of the wild-type promoter activity. After characterization of the promoter library employing a green fluorescent protein (GFP) variant, the new regulatory toolbox was successfully utilized in a ‘real case’, i.e. the expression of industrial enzymes. Characterization of the library under repressing, derepressing and inducing conditions displayed at least 12 cis-acting elements involved in PAOX1-driven high-level expression. Based on this deletion analysis, novel short artificial promoter variants were constructed by combining cis-acting elements with basal promoter. In addition to improving yields and quality of heterologous protein production, the new PAOX1 synthetic promoter library constitutes a basic toolbox to fine-tune gene expression in metabolic engineering and sequential induction of protein expression in synthetic biology.