Application of the YebF secretion pathway in Escherichia coli for rapid, on-plate screening of PETase libraries for...

Plastic waste such as polyethylene terephthalate (PET) is a major environmental burden, and enzymes capable of degrading PET are emerging as biocatalytic tools for sustainable recycling. Progress in improving PET hydrolases (or PETases) has been constrained by the lack of simple and reliable screening systems. Here, we report a functional screen for PETase activity in Escherichia coli based on a zone-clearing assay, where the YebF secretory pathway is used to secrete YebF-PETase onto agar plates supplemented with bis(2-hydroxyethyl) terephthalate (BHET). Enzyme activity is observed as zones of clearance around E. coli colonies that express active YebF-PETases, as insoluble BHET is converted to soluble products. As proof of concept, the screen was used to evaluated libraries of YebF-leaf-branch compost cutinase (LCC) generated by site-saturation mutagenesis at the active site residues Y95, L102, and V212. This led to the identification of the more active LCC variants V212T and L102F-V212T. The secretion-based assay was then validated using turbidity assays and untagged LCC variants, where the L102F-V212T variant was confirmed to be more active against PET than the wild-type enzyme. Docking simulations indicated that V212T improves substrate positioning in the active site while L102F modifies surface charge and hydrophobicity, potentially enhancing binding to the hydrophobic substrate. Overall, the YebF secretion-driven functional screen serves as a straightforward platform for identifying improved PETase variants and potentially other plastic degrading enzymes.