Effect of gene dosage and incubation temperature on production of β-mannanase by recombinant Pichia pastoris

High-level expression of -mannanase has been reported in Pichia pastoris under control of the GAP promoter. Two factors that strongly influence protein production and fermentation process development in Pichia pastoris protein expression system are gene dosage and cultivation temperature. The aim of this research was to improve the expression level of -mannanase in Pichia pastoris by proper increasing the gene dosage and decreasing the culture temperature. To this end, a panel of strains harboring different copy numbers of -mannanase gene were obtained by multiple zeocin concentration gradients screening, the influence of gene copy number on the expression of -mannanase in Pichia pastoris X33 was investigated. With the constitutive GAP promoter, the four copies strain exhibited a 4.04-fold higher -mannanase yield and a 1.83-fold higher total secretion proteins than the one copy strain, but an increase of the copy number above four resulted in a decrease of expression. Furthermore, the effects of culture temperature were studied in flask. The decreased culture temperature of four copies strain resulted in a 1.8-fold (26 degrees C) and 3.5-fold (22 degrees C) higher -mannanase activity compared to that at 30 degrees C. A fed-batch strategy was successfully used for high cell-density fermentation and -mannanase activity reached 2124 U/mL after cultivation for 72 h in a 5 L fermenter.