[Isolation and characterization of PAOX2 mutant in Pichia pastoris]

Spontaneous Mut+ mutants of P. pastoris AOX1-defective expression strain have been isolated, they were identified as phenotypically utilized methanol to grow as wild type. The results obtained from measuring growth curve when cultivated in medium in which methanol as a sole carbon source and detecting HSA protein on SDS-PAGE confirmed that the mutants have increased ability to utilize methanol and express foreign HSA gene product. The promoter region of AOX2 gene from the mutants has been cloned by PCR amplification, and the DNA fragment is 1022bp in size. Sequencing analysis showed that there are two point mutations at positions of -529 and -255 from the translation initiation codon respectively. The mutations improved AOX-1 defective function and facilitate the foreign gene for higher expression.