Phytase is an additive in animal feed that degrades phytic acid in plant material, reducing feeding costs, and pollution from fecal phosphorus excretion. A multistrategy approach was adopted to improve the expression of E. coli phytase in Pichia pastoris. We determined that the most suitable signal peptide for phytase secretion was an α-factor secretion signal with an initial enzyme activity of 153.51 U/mL. Increasing the copy number of this gene to four increased phytase enzyme activity by 234.35%. PDI overexpression and Pep4 gene knockout increased extracellular phytase production by 35.33% and 26.64%, respectively. By combining favorable factors affecting phytase expression and secretion, the enzyme activity of the phytase-engineered strain was amplified 384.60% compared with that of the original strain. We also evaluated the potential for the industrial production of the engineered strain using a 50-L fed-batch fermenter and achieved a total activity of 30,246 U/mL after 180 h of fermentation.
Keywords: Heterologous expression; Phytase; Pichia pastoris; Secretion.