The positive and negative cis-acting elements for methanol regulation in the Pichia pastoris AOX2 gene.

The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2. The AOX2 gene is transcribed at a much lower level than the AOX1 gene. Apart from this difference in expression levels, the two genes are regulated similarly. To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives. By analyzing the expression of HSA in P. pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter. The positive cis-acting element AOX2-UAS, located between positions -337 and -313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter. A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast. Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter. The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.