During the last decade, the methylotrophic yeast, Pichia pastoris, has become a major eukaryotic host for recombinant protein production in both academic and industrial research. Up to now, the expression of more than 500 proteins has been reported. One major reason for the success of this yeast as an expression system is the inducible promoter of its alcohol oxidase I (AOX1) gene. Its key features include an exceptional expression strength as well as a very strong glucose repression.

By computational sequence analysis several putative cis-acting elements could be identified within the AOX1 promoter sequence. Based on this sequence analyses, we performed deletion studies and identified both, positively and negatively acting promoter elements. Consequently, these elements were 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 regulation patterns.