Extracellular expression of alkaline phytase in Pichia pastoris: Influence of signal peptides, promoters and growth medium

Alkaline phytase isolated from pollen grains of Lilium longiflorum (LlALP) possesses unique catalytic and thermal stability properties that suggest it has the potential to be used as a feed supplement. However, substantial amounts of active enzymes are needed for animal feed studies and endogenous levels of LlALP in lily pollen are too low to provide the required amounts. Active rLlALP2 (coded by LlAlp2, one of two isoforms of alkaline phytase cDNA identified in lily pollen) has been successfully expressed in intracellular compartments of Pichia pastoris, however enzyme yields have been modest (25–30 mg/L) and purification of the enzyme has been challenging. Expression of foreign proteins to the extracellular medium of P. pastoris greatly simplifies protein purification because low levels of endogenous proteins are secreted by the yeast. In this paper, we first describe the generation of P. pastoris strains that will secrete rLlALP2 to the extracellular medium. Data presented here indicates that deletion of native signal peptides at the N- and C-termini of rLlALP2 enhanced α-mating factor (α-MF)-driven secretion by four-fold; chicken egg white lysozyme signal peptide was ineffective in the extracellular secretion of rLlALP2. Second, we describe our efforts to increase expression levels by employing a constitutive promoter from the glyceraldehyde-3-phosphate dehydrogenase gene (P(GAP)) in place of the strong, tightly controlled promoter of alcohol oxidase 1 gene (P(AOX1)). P(GAP) enhanced the extracellular expression levels of rLlALP2 compared to P(AOX1). Finally, we report on the optimization of the culture medium to enhance yields of rLlALP2. The strength of P(GAP) varies depending on the carbon source available for cell growth; secreted expression of rLlALP2 was highest when glycerol was the carbon source. The addition of histidine and Triton X-100 also enhanced extracellular expression. Taken together, the employment of P(GAP) under optimized culture conditions resulted in approximately eight-fold (75–80 mg/L) increase in extracellular activity compared to P(AOXI) (8–10 mg/L). The P. pastoris expression system can be employed as a source of active alkaline phytase for animal feed studies.