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

## TL;DR

Scientists engineered E. coli to break down PET plastic powder by redesigning a membrane protein with a catalytic triad, enabling the bacteria to grow on PET as a carbon source.

## Contribution

A novel computational workflow for designing catalytic triads in membrane proteins to enable PET hydrolysis without introducing foreign PETase genes.

## Key findings

- An engineered E. coli strain with a modified OmpA protein released 157 μM hydrolysis products from PET powder in 24 hours.
- The strain sustained growth on PET powder at a rate of 0.18 h⁻¹ at 37°C.
- Four surface-exposed proteins were successfully reprogrammed for PET hydrolysis using the new workflow.

## Abstract

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 Escherichia coli 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 E. coli strains for PET powder biodegradation without exogenous PETase genes.

•Triad exploration designs Ser-His-Asp/Glu catalytic triads across protein surfaces•Four secreted or surface-exposed proteins were recoded for PET hydrolysis•OmpA gene editing added a surface triad enabling PET powder hydrolysis by E. coli•Engineered strain released 157 μM products and grew (0.18 h−1) on PET powder

Triad exploration designs Ser-His-Asp/Glu catalytic triads across protein surfaces

Four secreted or surface-exposed proteins were recoded for PET hydrolysis

OmpA gene editing added a surface triad enabling PET powder hydrolysis by E. coli

Engineered strain released 157 μM products and grew (0.18 h−1) on PET powder

Computational molecular modeling; Molecular dynamics; Biological sciences; Protein; Bioengineering; Metabolic engineering; Synthetic biology