Amornloetwattana_2026_ChemSusChem_19_e202502560

Recent advances in biocatalytic recycling of polyethylene terephthalate (PET) using PET hydrolase enzymes have sparked interest in integrating PET degradation capabilities into living systems. Although cell-based strategies are limited by the mesophilic temperature constraints of microbial hosts, they offer a unique opportunity to couple PET depolymerization with biological upcycling into value-added chemicals. Here, a comprehensive approach for the cellular degradation and valorization of PET is reported. The crystal structure of MG8, a PET hydrolase identified from the human saliva metagenome is solved, and molecular dynamics simulations are used to pinpoint loop regions for targeted mutagenesis aimed at enhancing activity under moderate temperatures. Over 1000 MG8 loop variants are evaluated with a high-throughput mass spectrometric screening platform. Two catalytically improved mutants-MG8(G127Y/F250A) and MG8(N125S/G127Y/F250A)-exhibit significantly enhanced PET hydrolysis at 37 degreesC. To enable whole-cell PET valorization, a two-strain Escherichia coli system called PETCAT is constructed: one strain is engineered to secrete MG8(G127Y/F250A) for PET degradation, and the other harbors a synthetic pathway comprising seven heterologous genes for the conversion of terephthalic acid (TPA) into catechol, a versatile intermediate used in pharmaceuticals and fragrances. This study establishes a modular, one-pot microbial platform for PET recycling and upcycling under physiologically relevant conditions.