Novel genomically engineered antibiotic-free whole-cell biocatalysts for PET hydrolysis and waste remediation

Plastic pollution is an escalating environmental challenge requiring stable and sustainable biotechnological solutions. Here, we report a major advance in microbial PET degradation through the stable, inducer- and antibiotic-free whole-cell biocatalysts by genomic integration of PETase genes in Escherichia coli. Using a CRISPR-associated transposase system, PETase genes were inserted into defined chromosomal loci under a constitutive promoter, enabling continuous enzyme expression lacking plasmids or selection markers. Within this genomic framework, the Braun lipoprotein signal peptide (Lpp-SP) system was optimized to anchor and expose PETases efficiently to the bacterial surface, creating robust whole-cell biocatalysts. Surface localization and activity of the PETases were confirmed by biochemical assays, Western blotting, and flow cytometry, demonstrating robust surface exposure and sustained functionality across multiple generations. The displayed enzymes retained more than 90 % activity in EC-PET1 after 17 passages and approximately 70 % activity in EC-FP1 after 10 passages. Degradation byproducts, including mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA), were identified using high-performance liquid chromatography (HPLC), with optimized strain E. coli EC-FP1 producing 0.18 mM MHET production from semi-crystalline PET particles over 3 days using OD₆₀₀ = 1.0/mL. These results establish a proof-of-concept for genomically engineered microbes as robust and sustainable tools to address plastic waste at scale, offering long-term functional stability without the genetic instability and metabolic burden typical of plasmid-based systems.