Computationally guided genome rewiring of Escherichia coli and its application for nanopolyethylene terephthalate (PET) biodegradation and upcycling

Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.