Engineering Escherichia coli for polyethylene terephthalate powder biodegradation via recoding of an outer membrane protein

Deep computationally guided protein design enabled the introduction of a Ser-His-Asp catalytic triad that supports polyethylene terephthalate (PET) hydrolysis within an inner membrane <i>Escherichia coli</i> protein. This allows the engineering, through gene editing, of a strain capable of degrading PET particles smaller than 4.5 nm. We extended this approach to PET powder (<300 μm) using a computational workflow that builds geometrically pre-organized catalytic triads while preserving substrate binding in extracellular or surface-exposed membrane proteins. Four additional proteins were reprogrammed to degrade PET. Replacement of the outer membrane protein OmpA with a PETase-active variant carrying a surface-exposed artificial triad enabled an engineered strain to release 157 ± 2 μM hydrolysis products within 24 h at 37°C and to sustain growth (0.18 ± 0.07 h^-1) on PET powder as a carbon source. These results demonstrate the feasibility of engineering <i>E</i>. <i>coli</i> strains for PET powder biodegradation without exogenous PETase genes.