Biofilm Engineering for Efficient Substrate-Enzyme Interactions to Enhance Enzymatic PET Degradation

Despite extensive protein engineering efforts to significantly increase the catalytic efficiency of plastic-degrading enzymes, inefficient plastic-enzyme interactions during the interfacial biocatalytic process continue to impede their widespread applications. Here, we engineered the complete csgBACEFG curli operon through multicopy chromosomal integration to generate a csgBACEFG-optimized (CSGO) biofilm with enhanced curli assembly and plastic-surface adhesion. Unlike previou curli-based enzyme immobilization studies using individual CsgA curli components or pre-existing curli scaffolds, our strategy uses the complete curli assembly machinery to construct a dense, surface-associated catalytic biofilm. FAST-PETase was then embedded in a 2-day PET-surface CSGO biofilm through the SpyTag/SpyCatcher system, producing 2.5-fold more hydrolysis products from PET film depolymerization than soluble FAST-PETase with the same initial enzyme mass. The carrier-free biofilm-supported FAST-PETase also retained more than 50% of its first-cycle product formation after five 24-h PET nanoplastic (NanoPET) degradation cycles. These findings support biofilm-mediated enhancement of PET depolymerization and provide a proof-of-concept strategy that may inform future biofilm-assisted plastic treatment.