Spontaneous revertants of the AOX1 defective P.pastoris strain have been isolated,which were identified as phenotypically utilize methanol to grow as the wild type.The promoter region of the AOX2 gene from the revertant has been obtained by PCR amplification,and the DNA fragment is 1022 base pair in size.By the analysis of sequencing result and compared with the AOX2 gene sequences recorded in Gene Bank,two point mutations which at positions of 529 and 255(relative to the translation initiation codon)respectively,have been found.Since the positions are located in the AOX2 upstream respression sequences,the mutations may act interfere the repressor to combine with the functional sequences,and increase transcriptional activity.The above result implicated that the P.pastoris’ ability to utilize methanol could be incresed through modification the upper stream transcriptional regulation region in the AOX2 gene.
