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

TY - JOUR

T1 - Customization of Ethylene Glycol (EG)-Induced BmoR-Based Biosensor for the Directed Evolution of PET Degrading Enzymes

AU - Li, Min

AU - Chen, Zhenya

AU - Zhang, Wuyuan

AU - Wu, Tong

AU - Qi, Qingsheng

AU - Huo, Yi Xin

N1 - Publisher Copyright: © 2025 The Author(s). Advanced Science published by Wiley-VCH GmbH.

PY - 2025/4/3

Y1 - 2025/4/3

N2 - 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 SMM3F mutant demonstrates 1.59-fold higher terephthalic acid (TPA) production, with a 1.18-fold decrease in Km, a 1.29-fold increase in Vmax, and a 1.52-fold increase in kcat/Km, indicating stronger affinity and catalytic activity toward MHET. Furthermore, the SMM3F 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.

AB - 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 SMM3F mutant demonstrates 1.59-fold higher terephthalic acid (TPA) production, with a 1.18-fold decrease in Km, a 1.29-fold increase in Vmax, and a 1.52-fold increase in kcat/Km, indicating stronger affinity and catalytic activity toward MHET. Furthermore, the SMM3F 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.

KW - PET biodegradation

KW - biosensors

KW - directed evolution

KW - high-throughput screening

KW - value-added bioconversion

UR - https://www.scopus.com/pages/publications/105001710735

U2 - 10.1002/advs.202413205

DO - 10.1002/advs.202413205

M3 - Article

AN - SCOPUS:105001710735

SN - 2198-3844

VL - 12

JO - Advanced Science

JF - Advanced Science

IS - 13

M1 - 2413205

ER -