A multistrategy approach for improving the expression ofE. coliphytase inPichia pastoris

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 ofE. coliphytase inPichia pastoris. We determined that the most suitable signal peptide for phytase secretion was an alpha-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.