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Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5–9.0 and temperatures from 30–70 °C. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction. Enzymes have potential for recycling plastics such as PET, a polyester used in textiles and single-use packaging. Here, the authors identify and characterize additional PET-active biocatalysts and expand the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction. [ABSTRACT FROM AUTHOR]

Invited for this month′s cover is the BOTTLE Consortium, featuring Gregg Beckham's laboratory from NREL and John McGeehan's laboratory from the University of Portsmouth. The cover image shows the application of poly(ethylene terephthalate) (PET) hydrolase enzymes on post‐consumer waste plastic, towards the development of an enzymatic PET recycling strategy. The Full Paper itself is available at10.1002/cssc.202101932. [ABSTRACT FROM AUTHOR]

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2 II A3 II B II A1 II from Comamonas and tpaK from Rhodococcus jostii , (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis , and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET. • Enabled terephthalate (TPA) and bis(2-hydroxyethyl)TPA (BHET) catabolism in P. putida • Engineered conversion of BHET to β-ketoadipate (βKA), a performance-advantaged bioproduct • Achieved a titer of 15.1 g βKA/L at a 76% molar yield from commercial BHET • Demonstrated βKA production from glycolyzed poly(ethylene TPA) (PET) [ABSTRACT FROM AUTHOR]