Development of a self-assembled dual-enzyme co-display platform on the surface of the natural "chitosan beads" of yeast spores

Under starvation conditions, Saccharomyces cerevisiae diploid cells initiate meiosis to produce dormant cells called spores. When the DIT1 gene involved in assembling the outermost layer dityrosine is disrupted, the natural "chitosan beads" of yeast spores will be formed. A novel cell surface display system based on "chitosan beads" of dit1Δ yeast spores was previously established. In this study, a self-assembled dual-enzyme co-display platform on the surface of "chitosan beads" of S. cerevisiae spores was developed through the SpyTag/SpyCatcher system. As an example, two polyethylene terephthalate (PET) hydrolases, FAST-PETase (FPETase) and MHETase were self-assembled on the surface of spores. Compared with the unassembled enzymes, the assembled enzymes exhibited higher activity toward bis-hydroxyethyl terephthalate (BHET), achieving the complete degrading of 2 mM BHET within 1 h. Furthermore, the assembly of PETase and MHETase on the surface of spores demonstrated better thermostability (more than 85 % of initial activity after incubation at 30-70 °C for 12 h) and pH tolerance (approximately 80 % of original activity after incubation at pH 5.0 to 9.0 for 12 h). This study provides a novel and practical platform for the co-display and assembly of enzymes, offering a long-term stable enzyme catalyst for multi-enzyme cascade reactions especially conducted in complex environments.