Structure-guided extremophile genome mining expands the PETase landscape and reveals PET-degrading true lipase lineages

Poly(ethylene terephthalate) (PET) hydrolases are enzymes primarily within the polyesterase–cutinase branch of the α/β-hydrolase superfamily, whereas the contribution of true lipases to PET hydrolysis remains poorly explored. Here, we report the genome mining results of 18,082 extremophilic microorganisms, combined with structural modelling, enzyme–substrate simulations, and experimental validation, which enables the identification of PET-active true lipases. Two true lipases, LipBv and LipSh1, hydrolyse PET substrates, and homologous enzymes within their respective clusters also retain PET-hydrolysing activity, supporting the existence of specific lipase lineages associated with PET hydrolysis. Structural analyses suggest that the PETase-like activity and hydrolysis product profiles of these lipases are associated with differences in loop organization and active site accessibility. These lipase lineages clustered separately from 1,322 putative PETase-like hydrolases from the same extremophile genomes, while all groups remained distinct from previously characterized PETases. These findings expand the evolutionary diversity of PET-degrading enzymes in extremophiles. All files associated with this dataset can be downloaded directly from the Zenodo repository. Spreadsheet files can be opened with Microsoft Excel, LibreOffice Calc, or compatible software. Raw mass spectrometry data require MassLynx V4.1 (Waters) for processing. Network files can be imported into Cytoscape. File names and numbering correspond to the supplementary materials cited in the associated publication. Supplementary table 1. Reference and candidate true lipases identified during the genome mining workflow. This table summarizes the datasets used to identify candidate true lipases from extremophilic genomes. Reference lipase sequences were compiled and these sequences were used as queries for similarity searches against the Genome Taxonomy Database (GTDB) and other protein databases. Due to the size and complexity of the dataset, Supplementary table 1 is provided as a separate Microsoft Excel (.xlsx) file. All worksheets are labelled according to the workflow stages described in the manuscript and can be accessed using standard spreadsheet software. Supplementary table 2. Reference PETases and candidate PETase-like sequences identified from extremophilic genomes. This table summarizes the datasets used to identify putative PETase-like enzymes from extremophilic microbial genomes. Experimentally characterized PETase sequences were compiled and used as queries for homology-based searches against the GTDB. Owing to the large size of the dataset, Supplementary table 2 is provided as a separate Microsoft Excel (.xlsx) file. The file contains multiple worksheets corresponding to the different stages of the PETase mining workflow and can be viewed using standard spreadsheet software. Supplementary data 1. Raw MS data. The data in the raw folder can be analyzed with the software MassLynx V4.1 (Waters) to obtain the mass spectra of PET degradation oligomers. The mass spectrometry files can be analyzed with the software MassLynx V4.1. Supplementary data 2. Unprocessed HPLC data. Files are provided in .txt format corresponding to the data extracted with Varian ProStar software (Varian Inc., Palo Alto, California, USA). Supplementary data 3. Network. Net file that reconstructs the network. The network file is provided in .net format and can be directly imported into Cytoscape (version 3.0 or later recommended) for visualization and further network analysis. Supplementary data 4. Raw data for in vitro tests. Raw and processed datasets are provided in spreadsheet format, including replicate measurements and calculated values used for data analysis. These files enable full reproduction of the enzymatic activity calculations and figures reported in the manuscript.