Abstract
Poly(ethylene terephthalate) (PET) is the world’s most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer. Recently, a PETase enzyme from the bacterium Ideonella sakaiensis was shown to facilitate PET biodegradation, albeit at slow rate. Engineering of more efficient PETases is required for industrial relevance, but progress is currently hampered by the dependency on intracellular expression in Escherichia coli. To create a more efficient screening platform in E. coli, we explore different surface display anchors for fast and easy assaying of PETase activity. We show that PETases can be functionally displayed on the bacterial cell surface, enabling screening of enzyme activity on PET microparticles – both while anchored to the cell and following solubilization of the enzymes.
Highlights
Poly(ethylene terephthalate) (PET) is one of the most commonly produced plastics, appreciated for its low cost, robustness, and high durability
Selection of membrane anchors for IsPETase surface display in E. coli To overcome the bottleneck of inefficient heterologous expression and secretion of IsPETase variants in substantial amounts for screening and characterization, we adapted a surface display system previously described [14] to enable activity screening of IsPETase variants without the need for secretion into the growth medium
Two different membrane anchors were explored: (i) the C-terminal translocation unit of the Neisseria gonorrhoeae autotransporter IgA protease (C-IgAP) and (ii) a LppOmpA fusion protein consisting of the Lpp signal peptide followed by five transmembrane segments of E. coli outer membrane protein A (OmpA) (Fig. 1)
Summary
Poly(ethylene terephthalate) (PET) is one of the most commonly produced plastics (with an annual manufacturing of over 30 million tons), appreciated for its low cost, robustness, and high durability. Activity measurement on suspended PET Activity measurements of whole cell suspension (OD630 = 0.1) surface-displaying PETase variants and free PETase enzyme (post TEV cleavage, corresponding roughly to a concentration of 0.01–0.1 μM) on a suspended, semi-crystalline PET powder (Goodfellow Co, ES306031) were executed by adapting a plate readerbased assay previously described [17].
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