Franz S. Hartner1, Claudia Ruth2, Manuel Peter2, Andrea Mellitzer2, Bettina Leber2, Bettina Janesch3, Sandra Abad3, Kerstin Kitz3, and Anton Glieder4. (1) Research Centre Applied Biocatalysis, Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria, (2) Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria, (3) Research Centre Applied Biocatalysis, Petersgasse 14/2, 8010 Graz, Austria, (4) Institute of Molecular Biotechnology, Research Centre Applied Biocatalysis, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria
During the last decade, the methylotrophic yeast, Pichia pastoris, became a major eukaryotic host for recombinant protein production in both academic and industrial research. One major reason for the success of this yeast as an expression system is the strong and tightly regulated AOX1 promoter. Its key features include an exceptional expression strength as well as a very strong glucose repression. Although Pichia pastoris is used by many laboratories worldwide, there is still no way for tunable regulation of protein expression as needed for metabolic engineering and expression of difficult target proteins.
Within the AOX1 promoter sequence several putative cis-acting elements could be identified by computational sequence analysis. Based on this sequence analyses, we performed deletion studies and identified both, positively and negatively acting promoter elements. Consequently, these elements were individually tested by adding them to basal promoter elements and finally they were rearranged to generate synthetic and hybrid promoter libraries with different expression levels and regulatory profiles. Splitting and rearrangement of AOX1 promoter fragments resulted in a “one-for-all” promoter system. This particular promoter library can be used to adapt the time and level of transcript production for recombinant protein production and moreover represents a novel toolbox for fine-tuning the genetic control of genes used for metabolic engineering. Besides a library characterisation using fluorescent protein variants, the advantages of these new synthetic promoters for protein production and biocatalytic applications will be demonstrated.
