Construction of an episomal gap promoter-driven vector for surface display of fusion phytase in Saccharomyces cerevisiae

Abstract

Phytases are widely used as feed additives to improve phosphorus utilization and reduce phosphorus excretion in animal production systems. However, the catalytic performance of many phytases is restricted by pH fluctuations encountered throughout the gastrointestinal tract. To address this limitation, a fusion phytase comprising acid phytase (PhyE) and alkaline phytase (PhyB) was displayed on the cell surface of Saccharomyces cerevisiae with the aim of broadening its functional pH range. In addition, a strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (GAP promoter) was employed in an episomal yeast surface-display system, and two secretion signal peptides, Aga2P and MFα, were evaluated for their effects on phytase expression. Two recombinant strains, BY4743/pGAP-aGEB and BY4743/pGAP-mGEB, corresponding to the Aga2P and MFα signal peptides, respectively, were successfully constructed. PCR analysis confirmed the presence of the recombinant expression cassettes, while immunofluorescence staining verified successful surface localization of the fusion phytase. Functional analysis demonstrated that both recombinant strains exhibited significant phytase activity compared with the empty-vector control. At pH 5.0, the engineered strains released 297.3 ± 25.3 and 211.3 ± 5.8 nmol/mL inorganic phosphate, respectively, significantly exceeding the control strain (32.7 ± 2.1 nmol/mL). Phytase activity was also detected under alkaline conditions (pH 7.5), indicating that the fusion enzyme retained catalytic function across both acidic and alkaline pH ranges. Under acidic conditions, the Aga2P-mediated construct exhibited approximately 1.4-fold higher phosphate release than the MFα-mediated construct. Phosphate release levels under GAP promoter control were comparable to those previously reported for the TEF1-driven system. These findings demonstrate that the GAP promoter is an effective alternative for episomal yeast surface display of fusion phytase and that secretion signal optimization can further enhance expression efficiency. The developed system provides a promising platform for the production of whole-cell biocatalysts and feed enzyme applications.