Abstract
The immense volume of plastic waste poses continuous threats to the ecosystem and human health. Despite substantial efforts to enhance the catalytic activity, robustness, expression, and tolerance of plastic-degrading enzymes, the lack of high-throughput screening (HTS) tools hinders efficient enzyme engineering for industrial applications. Herein, we develop a novel fluorescence-based HTS tool for evolving polyethylene terephthalate (PET) degrading enzymes by constructing an engineered BmoR-based biosensor targeting the PET breakdown product, ethylene glycol (EG). The EG-responsive biosensors, with notably enhanced dynamic range and operation range, are customized by fluorescence-activated cell sorting (FACS)-assisted transcription factor engineering. The ingeniously designed SUMO-MHETase-FastPETase (SMF) chimera successfully addresses the functional soluble expression of MHETase in Escherichia coli and mitigates the inhibitory effect of mono-(2-hydroxyethyl) terephthalic acid (MHET) intermediate commonly observed with PETase alone. The obtained SM(M3)F mutant demonstrates 1.59-fold higher terephthalic acid (TPA) production, with a 1.18-fold decrease in K(m), a 1.29-fold increase in V(max), and a 1.52-fold increase in k(cat)/K(m), indicating stronger affinity and catalytic activity toward MHET. Furthermore, the SM(M3)F crude extract depolymerizes 5 g L(-1) bis-(2-hydroxyethyl) terephthalic acid (BHET) into TPA completely at 37 °C within 10 h, which is then directedly converted into value-added protocatechuic acid (PCA) (997.16 mg L(-1)) and gallic acid (GA) (411.69 mg L(-1)) at 30 °C, establishing an eco-friendly 'PET-BHET-MHET-TPA-PCA-GA' upcycling route. This study provides a valuable HTS tool for screening large-scale PET and MHET hydrolases candidates or metagenomic libraries, and propels the complete biodegradation and upcycling of PET waste.