The Generation of Multicopy Recombinant Strains
The extremely high levels of alcohol oxidase produced from the native AOX1 gene
The extremely high levels of alcohol oxidase produced from the native AOX1 gene in Pichia pastoris (5–30% of cell protein on induction) suggested that single-copy AOX1 -promoter expression vectors would be sufficient for efficient foreign gene expression. Therefore, the first strategy adopted for generating recombinant strains was to replace the AOX1 gene with a single copy of the foreign gene expression cassette (transplacement), since this type of transformant is the most stable. Some of the earliest studies supported this strategy, e.g., expression of β-galactosidase or hepatitis B surface antigen was efficient and was not improved by increasing vector copy number (1 ,2 ).