A Novel Vector for Construction of Markerless Multicopy Overexpression Transformants in .

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/ 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/ recombination system for multiple integrations of target gene to construct multicopy expression strain of . P promoter was fused to to construct a methanol inducible Cre recombinase. The leakage expression of Cre recombinase in was blocked by introducing the operator gene . The expression vector designed pMCO-AOXα was stable in and could effectively rescue the Zeocin resistance gene for next round of integration in . Phytase AppA from was chosen as a reporter gene. Transformants with 2-16 copies of were constructed by using a single antibiotic. Expression of was gene dosage dependent when <12 copies were integrated. The protein yield increased 4.45-folds when 12 copies of were integrated comparing with the single copy integration. Our results showed that pMCO-AOXα was highly effective for rational construction of multicopy transformat in .