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bioRxiv · 10.1101/2024.11.20.624590

Antibiotic-free whole-cell biocatalytic fermentation: Escherichia coli with surface-displayed PETases for sustainable plastic degradation

Romero-Orejon, K.·Karbalaei-Heidari, H. R.·Budisa, N.·Levin, D.
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Abstract

Plastic pollution has increasingly burdened the environment, driving the need for natural degradation platforms that utilize microbial enzymes to break plastics down into monomers. In this study, we introduce a novel approach using Escherichia coli as a fermentative, antibiotic-free whole-cell biocatalyst with surface-displayed, genomically integrated PETases for efficient plastic degradation. PETases, a class of esterases, catalyze the hydrolysis of polyethylene terephthalate (PET) into mono-2-hydroxyethyl terephthalate (MHET). Surface display of these enzymes was achieved via gene fusions with an N-terminal cysteine (Cys) triacylated anchor, mediated by the Braun lipoprotein (Lpp) signal peptide. To circumvent issues associated with plasmids, - such as genetic instability and reliance on antibiotics - we used a Type I-F CRISPR-associated transposase to insert the genes directly into specific E. coli genome sites. Proper enzyme display and activity on the E. coli surface were confirmed through enzyme activity tests, Western blotting, and flow cytometry, with cells retaining PET degradation ability over multiple generations. High-performance liquid chromatography (HPLC) analysis assessed degradation efficiency, identifying byproducts such as bis (2-hydroxyethyl) terephthalate and terephthalic acid. This study establishes a proof-of-concept for efficient plastic degradation using engineered bacteria as robust, sustainable, and genomically stable whole-cell biocatalysts, providing a promising platform for addressing plastic waste management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/624590v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1320dd8org.highwire.dtl.DTLVardef@12b025forg.highwire.dtl.DTLVardef@a8b0b3org.highwire.dtl.DTLVardef@e8d2c7_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence AbstractEscherichia coli was engineered as a fermentative, antibiotic-free whole-cell biocatalyst, featuring surface-displayed and genomically integrated PETases for efficient plastic degradation. This innovative approach has the potential to transform plastic recycling by enabling sustainable, large-scale degradation of plastic waste through environmentally friendly microbial systems.

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Romero-Orejon, K., Karbalaei-Heidari, H. R., Budisa, N., Levin, D.. 2024-11-21. Antibiotic-free whole-cell biocatalytic fermentation: Escherichia coli with surface-displayed PETases for sustainable plastic degradation. https://doi.org/10.1101/2024.11.20.624590

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