The inability of non-ruminant animals to digest phytates in animal feed has contributed to phosphate pollution of soil and water bodies. Supplementation of animal feed with phytases, the enzyme that catalyzes the hydrolysis of phytic acid, has proven to be an effective strategy to alleviate environmental phosphate contamination and increase the bioavailability of phosphates to animals. The unique catalytic and thermal stability properties of alkaline phytase from lily pollen (LlALP) suggest that it has the potential to be used as a feed supplement. Substantial amounts of LlALP are needed for animal feed studies. Active rLlALP2 has been successfully expressed in Pichia pastoris, however, enzyme yields have been modest (25-30 mg/L) and rLlALP2 purification has been challenging. Purification is significantly simplified if proteins can be secreted to the extracellular medium. Secretion levels are dependent on the presence of an appropriate secretion signal upstream of the rLlAlp2 transgene and the influence that native signal peptide(s) present in rLlAlp2 have on secretion. Data presented here suggests that the native signal peptides at N- and C-termini of rLlAlp2 significantly decreased a-mating factor (a-MF)-driven secretion of rLlALP2 to the expression medium; deletion of native signal peptides at both the N- and C-termini of rLlALP2 enhanced secretion by four-fold. Chicken egg white lysozyme (CL) secretion signal peptide was ineffective in the extracellular secretion of rLlALP2. To enhance rLlALP2 secretion, the effectiveness of the strong inducible AOX1 promoter (PAOX1) was compared with the constitutive promoterfrom the glyceraldehyde-3-phosphate dehydrogenase gene (PGAP). The extracellular expression level of rLlALP2 under the control of PGAP was enhanced around eight-fold (75 mg/L) compared to P AOX1 (10 mg/L). The strength of PGAP varies depending on the carbon source used for cell growth; data presented here indicates that secreted expression of rLlALP2 was highest when glycerol was the carbon source.

Extracellular expression of alkaline phytase in<i> Pichia pastoris</i>: The GAP promoter is more efficient than the AOX1 promoter

246th National Meeting (2013)

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Extracellular expression of alkaline phytase in Pichia pastoris: The GAP promoter is more efficient than the AOX1 promoter