An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate

The study addresses whether archaeal enzymes can degrade polyethylene terephthalate (PET), a pervasive pollutant. Although numerous PET-degrading enzymes have been identified from bacteria and some fungi, no archaeal PETase had been reported. The authors searched metagenomic data for archaeal candidates and identified PET46 from a Candidatus Bathyarchaeota metagenome-assembled genome from the Guaymas Basin. The purpose was to characterize PET46 structurally and biochemically, test its activity on PET and PET-derived intermediates (BHET, MHET), and compare its properties with established bacterial PETases (IsPETase) and cutinases (LCC). Establishing archaeal PET depolymerization would expand known enzyme diversity and inform environmental and biotechnological strategies for PET degradation.

Previous work has cataloged ~200 polymer-active enzymes (PAYZ database), including PETases primarily from bacterial phyla (Actinobacterota, Pseudomonadota, Bacillota, Bacteroidota, Chloroflexota) and a few from fungi (e.g., CalB, HiC, Fusarium solani). Common PETase features include an α/β-hydrolase fold, a Ser-Asp-His catalytic triad, an exposed active site, and aromatic residues contributing to PET binding and the oxyanion hole. IsPETase and LCC are among the best characterized bacterial enzymes. PETase activity at lower temperatures has been demonstrated, although many highly active PETases are thermostable with optima 55–65 °C, supporting industrial relevance. Feruloyl esterases (FAEs), which release hydroxycinnamic acids from plant cell walls, share structural traits with PETases and have been proposed as evolutionary relatives; however, PET-degrading activity had not been established for archaeal enzymes before this work.

• Bioinformatic discovery: The authors used a profile Hidden Markov Model (HMM) search against NCBI's non-redundant database filtered for archaeal sequences (tax ID 2157) to identify putative PET-degrading hydrolases. PET46 was selected based on HMM bit score, sequence length, and initial analyses; sequence similarity networks were constructed to contextualize PET46 among known PETases and FAEs.
• Gene synthesis and expression: The PET46 gene was codon-optimized, synthesized into pET21(+), and expressed heterologously in Escherichia coli BL21(DE3). Mutants (including lid variants) were generated by site-directed mutagenesis. IsPETase and LCC were produced similarly with appropriate vectors.
• Protein purification: Cells were lysed (French Press) and proteins purified by Ni-NTA affinity chromatography, followed by dialysis into potassium phosphate buffer.
• Crystallography: PET46 was crystallized by sitting-drop vapor diffusion at 12 °C. Diffraction data were collected at ESRF, and the structure solved by molecular replacement with iterative model building (COOT) and refinement (Phenix). Data collection and refinement statistics report 1.71 Å resolution; the structure shows an α/β-hydrolase fold with a distinctive lid domain.
• Structural comparison: Structural alignments and similarity analyses (DALI, heatmaps) compared PET46 with bacterial PETases and FAEs. Key features in loops, catalytic triad, aromatic clamp, and disulfide bonds were analyzed.
• Substrate docking: AutoDock with DrugScore2018 was used to dock BHET into the PET46 active site using a Lamarckian genetic algorithm. Docking clusters and distances to the catalytic serine were assessed; potential oxyanion hole contributors and substrate-access residues were identified.
• PET and oligomer/monomer degradation assays: Activities on BHET, MHET, and 3PET (trimer) were quantified by UHPLC. PET degradation was assayed on semi-crystalline PET powder and amorphous foil at 30, 50, 60, and 70 °C in phosphate buffer, comparing PET46 to IsPETase and LCC (equal enzyme concentrations). Product formation (TPA, MHET, BHET) was measured over up to 72 h, correcting for autohydrolysis controls.
• Biochemical characterization: Substrate specificity was assessed with pNP-esters (C4–C18). Temperature and pH optima, thermal stability (nanoDSF), effects of metal ions (Ca2+, Co2+, Cu2+, Fe3+, Mg2+, Mn2+, Zn2+), detergents (Triton X-100), DTT, and organic solvents (acetone, DMF, DMSO, isopropanol) were measured. Stability over days at elevated temperatures was monitored.

• First archaeal PET-degrading enzyme: PET46, a feruloyl esterase from a Candidatus Bathyarchaeota MAG (Guaymas Basin), depolymerizes semi-crystalline PET and hydrolyzes BHET and MHET.
• Structure: Crystal structure solved at 1.71 Å (PDB accession 8ABU). PET46 adopts an α/β-hydrolase fold with the canonical Ser-His-Asp catalytic triad and a unique lid domain characteristic of FAEs. It differs from bacterial PETases by lacking the typical aromatic clamp composition and certain disulfide features near the active site.
• Lid importance: The lid domain participates in substrate binding and is crucial for activity; a lid variant (PET46Δ) could convert BHET to MHET but lost MHET hydrolysis, indicating a role in accommodating and processing intermediates.
• Docking insights: BHET docking produced two main pose clusters with catalytic distances (<3.1 Å) consistent with catalysis. Backbone amides of Phe39 and Met161 likely stabilize the transition state (oxyanion hole). Residues Ala46, Ala140, and Lys147 were implicated in substrate accessibility/binding.
• Activity on PET powder: At 60 °C, 3 μM PET46 released up to ~1.6 mM aromatic products (predominantly TPA >99%) from semi-crystalline PET powder in 200 μL after 72 h, corresponding to ~3.38% conversion (48 mM TPA equivalents). No activity was detected on PET foil.
• Comparison with bacterial enzymes: At their optima, PET46 yielded product levels comparable in magnitude to IsPETase and lower than LCC on PET powder, but PET46 outperformed both in converting intermediates BHET and MHET.
  • Specific activities (U mg−1): PET46 BHET 3.103; MHET 0.020; ratio BHET:MHET 155.85. LCC BHET 2.202; MHET 0.016 (ratio 139.87). IsPETase BHET 0.983; MHET 0.011 (ratio 85.78).
• Temperature and stability: PET46 shows broad temperature activity with an optimum around 70 °C; nanoDSF melting temperature Tm ≈ 85.45 °C. Retains >60% activity at 60 °C for up to 21 days; retains activity at 70 °C over days but with faster decay.
• pH profile and ions/solvents: Active from pH 5–8 (optimum pH 7–8). Zn2+ nearly doubled activity; Ca2+ reduced activity by ~50%. DTT increased activity (~2-fold). The enzyme tolerated organic solvents; 10% acetone and 5% DMF/DMSO increased activity (~2-fold).
• Substrate scope: Highest activity on pNP-decanoate (C10); lower activity on shorter (C4–C6) and longer (C12–C16/18) chains, consistent with esterase promiscuity.

The findings demonstrate that an archaeal feruloyl esterase (PET46) can depolymerize PET, addressing the gap that no archaeal PETase had been identified previously. Structural and functional data indicate PET46 shares the core PETase architecture but uniquely contains a lid domain, which governs substrate access and processing, explaining its efficacy on PET intermediates and polymer powder at elevated temperatures. This expands the known diversity of PET-hydrolyzing scaffolds beyond bacterial enzymes and supports the hypothesis that FAEs and related esterases can evolve PETase activity. The thermostability and solvent/ion tolerance of PET46 reflect adaptation to Guaymas Basin conditions and are advantageous for biotechnological applications. Environmentally, Bathyarchaeota are abundant in deep-sea sediments; PET46-like enzymes could contribute to slow PET turnover in the deep ocean, especially on microplastics, although rates are low. Comparisons to IsPETase and LCC show that while PET46 may be less efficient on bulk PET than optimized cutinases, it is more active on intermediates, making it a promising component in multi-enzyme depolymerization strategies. Structure-guided engineering targeting the lid and substrate-binding residues could further enhance PET46’s activity and stability.

This work reports PET46 as the first archaeal enzyme shown to depolymerize PET, structurally characterized at high resolution and biochemically validated. PET46 combines an α/β-hydrolase core with a feruloyl esterase-like lid critical for substrate engagement, exhibits thermostability (Tm ~85 °C) and broad pH tolerance, and efficiently hydrolyzes BHET and MHET, while degrading semi-crystalline PET powder at elevated temperatures. The results broaden the repertoire of PET-degrading scaffolds and suggest that archaeal FAEs may be a valuable source for PETase discovery and engineering. Future research should: (i) engineer the lid and substrate-binding residues to improve polymer binding and turnover; (ii) explore secretion and surface-binding modules to enhance PET contact; (iii) survey archaeal metagenomes for additional candidates; (iv) assess synergistic multi-enzyme systems; and (v) investigate environmental prevalence and in situ activity of PET46-like enzymes in deep-sea plastisphere communities.

• No activity was detected on amorphous PET foil, indicating substrate form and crystallinity constraints.
• Effective PET depolymerization required elevated temperatures (≥50–60 °C), limiting ambient applicability.
• Conversion on PET powder was modest (~3–4% under assay conditions), lower than optimized engineered cutinases.
• PET46 originates from a metagenome-assembled genome of a non-cultivated archaeon; in vivo expression, secretion, and physiological role remain to be validated. No obvious N-terminal secretion signal was identified.
• Some structural/comparative data rely on in silico docking and inference; co-crystal structures with PET-like ligands were not reported.