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Characterization and engineering of a two-enzyme system for plastics depolymerization

Research output: Contribution to journalArticlepeer-review

  • Brandon C. Knott
  • Erika Erickson
  • Mark Devin Allen
  • Japheth E. Gado
  • Rosie Graham
  • Fiona Kearns
  • Isabel Pardo
  • Ece Topuzlu
  • Jared Anderson
  • Harry Austin
  • Graham Dominick
  • Christopher W. Johnson
  • Nicholas A. Rorrer
  • Caralyn Szostkiewicz
  • Valérie Copié
  • Christina Payne
  • H. Lee Woodcock
  • Bryon S. Donohoe
  • Gregg T. Beckham
  • Professor John McGeehan
Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products including mono-(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono-(2-hydroxyethyl)-furanoate or mono-(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all non-zero MHETase concentrations tested. Lastly, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.
Original languageEnglish
Article number0
Pages (from-to)25476-25485
Number of pages10
JournalProceedings of the National Academy of Sciences of the United States of America
Volume117
Issue number41
Early online date28 Sep 2020
DOIs
Publication statusPublished - 13 Oct 2020

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