Customization of Ethylene Glycol (EG)‐Induced BmoR‐Based Biosensor for the Directed Evolution of PET Degrading Enzymes

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^M3F 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^M3F 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.