Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity

Erika Erickson, Japheth E. Gado, Luisana Avilan, Felicia Bratti, Richard K. Brizendine, Paul Cox, Raj Gill, Rosie Graham, Dong-Jin Kim, Gerhard Koenig, William E. Michener, Saroj Poudel, Kelsey J. Ramirez, Thomas Jack Shakespeare, Michael Zahn, Eric S. Boyd, Christina Payne, Jennifer L. DuBois, Andrew R. Pickford, Gregg T. Beckham*John McGeehan*

*Corresponding author for this work

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Abstract

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.
Original languageEnglish
Article number7850
Number of pages15
JournalNature Communications
Volume13
Issue number1
DOIs
Publication statusPublished - 21 Dec 2022

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