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Pichia pastoris  is widely used as a platform for heterologous protein expression because of its high volumetric productivity. Multicopy integration of the target gene is commonly used to improve the production of the target protein. Cre/ lox  recombination system is a powerful tool for the marker rescue during multiple integrations with one selection marker. Here we reported a novel expression vector based on the Cre/ lox  recombination system for multiple integrations of target gene to construct multicopy expression strain of  P. pastoris . P AOX1  promoter was fused to  cre  to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in  Escherichia coli  was blocked by introducing the operator gene  lacO . The expression vector designed pMCO-AOXα was stable in  E. coli  and could effectively rescue the Zeocin resistance gene for next round of integration in  P. pastoris . Phytase AppA from  E. coli  was chosen as a reporter gene. Transformants with 2–16 copies of  appA  were constructed by using a single antibiotic. Expression of  appA  was gene dosage dependent when <12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of  appA  were integrated comparing with the single copy integration. Our results showed that pMCO-AOXα was highly effective for rational construction of multicopy transformat in  P. pastoris .

A Novel Vector for Construction of Markerless Multicopy Overexpression Transformants in Pichia pastoris