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

作者：Min Li, Zhenya Chen, Wuyuan Zhang, Tong Wu, Qingsheng Qi, Yi‐Xin Huo · 发表于：Advanced Science · 年份：2025 · DOI：10.1002/advs.202413205 · 被引用次数：9 · 研究领域：Microplastics and Plastic Pollution、Graphene and Nanomaterials Applications、biodegradable polymer synthesis and properties

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 directedl...