Improving Phytase Expression by Increasing the Gene Copy Number of appA-m in Pichia pastoris

To improve the fermentation potency of phytase in recombinant hosts and decrease the production cost, the Pichia expression vector pGAPZα-A was modified by introduction of an AOX1 promoter to generate the plasmid, pAOXZα. A phytase gene appA-m was cloned into pAOXZα to construct a methanol-induced expression vector pAOXZα-appA-m. The recombinant Pichia pastoris 74#, which contained one copy of appA-m with a fermentation potency exceeding 7.5×106 IU/mL, was used as the host strain for the transformation of pAOXZα-appA-m. The P. pastoris transformants were obtained by electroporation. PCR results indicated that the newly introduced appA-m expression frame had integrated into the genome of P. pastoris and that the original construction of phytase gene still existed and had not changed. SDS-PAGE analysis revealed that phytase was overexpressed and was secreted into the culture supernatant. Recombinants with high expression level were screened and used for fermentation. In a 5-L fermenter, the expression level of phytase protein reached 4 mg/mL and the phytase activity (fermentation potency) exceeded 1.2×107 IU/mL, which was about 1.6-fold that of the host strain 74#. Moreover, the improved recombinant P. pastoris showed excellent expression stability and heredity stability.