Sourcing Thermotolerant Poly(ethylene terephthalate) Hydrolase Scaffolds from Natural Diversity

Erika Erickson, Japheth Gado, Luisana Avilan, Felicia Bratti, Richard Brizendine, Paul Cox, Raj Gill, Rosie Graham, Dong-Jin Kim, Gerhard Konig, William Michener, Saroj Poudel, Kelsey Ramirez, Thomas Shakespeare, Michael Zahn, Eric Boyd, Christina Payne, Jennifer DuBois, Andrew Pickford, Gregg BeckhamJohn McGeehan

Research output: Contribution to journalArticlepeer-review

53 Scopus Citations

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 languageAmerican English
Article numberArticle No. 7850
Number of pages15
JournalNature Communications
Volume13
Issue number1
DOIs
StatePublished - Dec 2022

Bibliographical note

Publisher Copyright:
© 2022, The Author(s).

NREL Publication Number

  • NREL/JA-2A00-82013

Keywords

  • AlphaFold
  • esterase
  • interfacial biocatalysis
  • machine learning
  • PETase
  • plastics recycling
  • plastics upcycling

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