Polyethylene terephthalate (PET) pollution is a global challenge. Advancing the bioprospecting of PET‐degrading enzymes through metagenomics and using computational and functional methods to identify key positions influencing the catalytic rate and selectivity are part of the solution. Here, we report PETase activity in the metagenomic lipase Lip_MRD9, which exhibits peak activity at 30 °C and pH 9.0 and has a denaturation temperature of 42 °C. In addition to acting on long‐chain triglycerides (up to ∼13 units (U)/mg, pH 8.0, 30 °C) and a wide range of 34 other esters (up to ∼228 U/g), Lip_MRD9 hydrolyses mono(2‐hydroxyethyl) terephthalate (∼57 U/g) and bis(2‐hydroxyethyl) terephthalate (∼131 U/g). It also efficiently deconstructs GoodFellow amorphous submicro‐ and nanosized PET particles (∼984/2238 μM degradation products at 30/40 °C, pH 7.0, 21.5 h) and films (∼112/198 μM degradation products at 30/40 °C, pH 7.0, 7 days). Through molecular modelling and experimental analysis, the active site of Lip_MRD9 was revealed, identifying a key residue contributing to its PETase activity compared with those of its closest homologues. This residue plays a crucial role in determining the distinct profiles of degradation products from PET hydrolysis and should be studied in other PETases for its influence on the catalytic process.

Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.

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.

Lubricants based on fatty acid ester (FAE) mixtures are widely used in the textile industry, e.g., in spin finishes applied during the production of synthetic fibres, or in sizes added to fibres before weaving. FAE lubricants can significantly impact the dyeing quality of a textile due to their hydrophobicity and must therefore be removed before dyeing. However, the solvents currently used for their removal pose an environmental risk, and biobased solutions are thus sought. A lipase-assisted pre-dyeing treatment for synthetic fibre textiles was developed in this study. Six lipases were tested for their ability to hydrolyse FAEs from a polyamide-with-elastane textile, and all were found to be active. The conditions for the washing of lipase-treated textiles were found to be crucial for the performance of the process. Among the possible lipid hydrolysis products of tripalmitin (selected as a model FAE), only palmitic acid removal improved during washing, in comparison with the original FAE. This improvement only occurred with washing solutions containing a monovalent base. A combination of lipase treatment and washing with a non-ionic surfactant and monovalent base was found to be effective in the removal of FAEs, with a performance similar to a current solvent-based pre-treatment process.

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, Lip _Bv and Lip _Sh1 , 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 differences in loop organization and active‐site accessibility may contribute to the observed PET‐hydrolysing activity and hydrolysis product profiles. These lipase lineages clustered separately from 1322 putative PETase homologues from the same extremophile genomes, while all groups remained distinct from previously characterized PETases. These findings expand the evolutionary diversity of PET‐hydrolysing enzymes in extremophiles.

El libro colectivo que a continuación se ofrece al lector es producto del trabajo realizado entre los años 2016 y 2018, en el contexto del proyecto de investiga...