Open AccessAn upstream activation sequence controls the expression of AOX1 gene in Pichia pastoris
Author(s) -
Xuan Yaoji,
Zhou Xiangshan,
Zhang Wenwen,
Zhang Xiao,
Song Zhiwei,
Zhang Yuanxing
Publication year - 2009
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2009.00571.x
Subject(s) - biology , pichia pastoris , alcohol oxidase , promoter , gene , microbiology and biotechnology , alcohol dehydrogenase , saccharomyces cerevisiae , transcription (linguistics) , upstream activating sequence , mutant , gene expression , regulatory sequence , plasmid , electrophoretic mobility shift assay , genetics , recombinant dna , biochemistry , enzyme , linguistics , philosophy
Alcohol oxidase I gene ( AOX1 ) promoter (P AOX1 ) is a key promoter in the methylotrophic yeast Pichia pastoris . To identify the cis ‐acting element in the AOX1 promoter, we constructed expression plasmids in which the green fluorescent protein (GFP) gene coding region was fused to a series of internal deletion mutants of the AOX1 promoter. By analyzing the expression and transcription level of GFP by each plasmid, we identified a positive cis ‐element, Region D, which is located between positions −638 and −510 of the AOX1 promoter. This region contains an invert repeat‐like sequence GTGGGG TCAAATAGTTTCATGTT CCCCAA that is similar to the upstream activation sequence 1 (UAS1) of alcohol dehydrogenase II gene ( ADH2 ) in Saccharomyces cerevisiae . The inverted repeat sequence in the UAS1 is known to contain the binding site for alcohol dehydrogenase II synthesis regulator (Adr1p). When three tandem copies of Region D were inserted into the Region D‐deleted AOX1 promoter, the expression of GFP at the protein level and the mRNA level increased to 157% and 135% of the wild type, respectively. An electrophoretic mobility shift assay indicated that Region D could form a DNA–protein complex with cell extracts under methanol‐induced and glucose/methanol‐repressed conditions. These data suggest that Region D may function as a cis ‐acting regulatory element in the AOX1 promoter to positively regulate the expression of AOX1 .