The extremely high levels of alcohol oxidase produced from the nativeAOX1gene inPichia pastoris(5–30% of cell protein on induction) suggested that single-copyAOX1-promoter expression vectors would be sufficient for efficient foreign gene expression. Therefore, the first strategy adopted for generating recombinant strains was to replace theAOX1gene 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 ).
