Our goal is to develop a whole-cell biocatalyst by displaying PETase and MHETase on the surface of yeast cell (Candida Tropicalis) to degrade PET waste. Firstly, we chose a robust Candida Tropicalis as the host and deleted the URA3 gene of genome to block the uracil synthesis by CRISPR-Cas9. In order to display the protein more efficiently, we must obtain the appropriate anchor protein. Therefore, we built a model to predict the anchor protein candidates. We used an enhanced green fluorescent protein (eGFP) as the reporter and inserted the codon-optimized GFP gene to the genome of Candida Tropicalis by homologous recombination. Finally, we selected the best one from 89 anchor proteins, fused it with PETase, and then incubated the yeast with PET waste. We hope the yeast with surfaced PETase can degrade the PET more efficiently and achieved the recycling of PET waste.
