Development of CRISPR/Cas9-Mediated Saccharomyces cerevisiae Strains for the Cell-Surface Display 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 alpha-agglutinin-GPI system in two marker-free strains, BY4743::GAP-mGEB and BY4743::GAP-aGEB, employing MF alpha 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 K-m of 0.377-0.989 mM and a k(cat) of 0.014-0.019 mu mol/min/mg wet-cell weight, with the Aga2p strain showing the highest efficiency. The fusion phytase exhibited similar to 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.