Development ofCRISPR/Cas9-Mediated Saccharomycescerevisiae Strains for the Cell-SurfaceDisplay of a Novel Fusion Acid-Alkaline Phytase

Phytases enhance phosphorus bioavailability in animal feed, but their limited reusability hinders their application. To overcome this, Saccharomyces cerevisiae was engineered to display a fusion phytase combining acid and alkaline phytases on its cell surface by using CRISPR/Cas9. The enzyme was anchored via the α-agglutinin-GPI system in two marker-free strains, BY4743::<i>GAP-mGEB</i> and BY4743::<i>GAP-aGEB</i>, employing MFα and Aga2p signal peptides, respectively. Both strains exhibited robust surface activity across a broad pH range, retaining >50% relative activity between pH 1.0 and 7.0, with dual optima at pH 2.0 and 5.0–6.0. Kinetic analysis revealed a <i>K</i>_m of 0.377–0.989 mM and a <i>k</i>_cat of 0.014–0.019 μmol/min/mg wet-cell weight, with the Aga2p strain showing the highest efficiency. The fusion phytase exhibited ∼ 3.5–4 times higher activity than the single acid phytase. These strains effectively degraded phytate in soybean, corn flour, and rice bran, demonstrating a sustainable approach for improving phosphorus utilization in animal feed.