(154f) Two Enzyme Whole Cell Biocatalysts for Complete PET Degradation

A promising solution to the challenge of polyethylene terephthalate (PET) upcycling for environmental sustainability is enzymatic degradation.¹ Although much effort has been made towards efficient plastic degradation through protein engineering² and whole cell surface display³, a systematic approach using an engineered bacterial community for complete PET degradation is still required. The hypothesis is that a bacterial community with surface displayed PET-degrading enzymes will completely degrade PET to its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG). Whole cell biocatalysts displaying PET-degrading enzymes FAST-PETase² and MHETase⁴ on the surface of Escherichia coli (E. coli) were constructed. Additionally, high throughput assays including bis(2-hydroxyethyl) terephthalate (BHET)-agar plates and fluorogenic fluorescein dibenzoate (FDBz)⁵ were explored.

FAST-PETase and MHETase were surface displayed on E. coli using transporter proteins YeeJ³ and AIDA-I⁶ because of their demonstrated ability to efficiently surface display heterologous passenger proteins.⁷ These whole-cell biocatalysts eliminate the need for costly enzyme purification and enhance enzymatic activity. After expression, activity of surface displayed FAST-PETase was determined by incubating cells with PET films for one week at 25°C followed by HPLC analysis to detect degradation products. Although loss of cell viability within 48 hours of expression was observed for AIDA-I-FAST-PETase, PET degradation products were detected, demonstrating the active expression of FAST-PETase. A similar procedure utilizing dissolved MHET as reactant was performed to confirm the activity of both AIDA-I-MHETase and YeeJ-MHETase via HPLC.

High-throughput screening and kinetic assays for PETase were utilized for rapid screening of PET-degrading activity. BHET plates showed visual changes in both purified and whole-cell surface displayed FAST-PETase while none was detected for MHETase, as expected. Furthermore, a fluorogenic compound with PET-like ester bonds, FDBz, was used to test PETase activity. A linear correlation between fluorescence intensity and enzyme concentration was observed with purified enzymes.

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