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

Knott, Brandon C.; Erickson, Erika; Allen, Mark D.; Gado, Japheth E.; Graham, Rosie; Kearns, Fiona L.; Pardo, Isabel; Topuzlu, Ece; Anderson, Jared J.; Austin, Harry P.; Dominick, Graham; Johnson, Christopher W.; Rorrer, Nicholas A.; Szostkiewicz, Caralyn J.; Copié; , Valé; rie; Payne, Christina M.; Woodcock, H. Lee; Donohoe, Bryon S.; Beckham, Gregg T.; McGeehan, John E.

doi:10.1073/pnas.2006753117

[해외논문] Characterization and engineering of a two-enzyme system for plastics depolymerization 원문보기

Proceedings of the National Academy of Sciences of the United States of America, v.117 no.41, 2020년, pp.25476 - 25485

Knott, Brandon C. (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401) , Erickson, Erika , Allen, Mark D. (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401) , Gado, Japheth E. , Graham, Rosie (Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom) , Kearns, Fiona L. , Pardo, Isabel (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401) , Topuzlu, Ece , Anderson, Jared J. (Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom) , Austin, Harry P. , Dominick, Graham (Department of Chemistry, University of South Florida, Tampa, FL 33620) , Johnson, Christopher W. , Rorrer, Nicholas A. (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401) , Szostkiewicz, Caralyn J. , Copié, Valérie (Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401) , Payne, Christina M. , Woodcock, H. Lee (Renewable Resources and Enabling Scienc) , Donohoe, Bryon S. , Beckham, Gregg T. , McGeehan, John E.

Abstract ▼ AI-Helper

SignificanceDeconstruction of recalcitrant polymers, such as cellulose or chitin, is accomplished in nature by synergistic enzyme cocktails that evolved over millions of years. In these systems, soluble dimeric or oligomeric intermediates are typically released via interfacial biocatalysis, and additional enzymes often process the soluble intermediates into monomers for microbial uptake. The recent discovery of a two-enzyme system for polyethylene terephthalate (PET) deconstruction, which employs one enzyme to convert the polymer into soluble intermediates and another enzyme to produce the constituent PET monomers (MHETase), suggests that nature may be evolving similar deconstruction strategies for synthetic plastics. This study on the characterization of the MHETase enzyme and synergy of the two-enzyme PET depolymerization system may inform enzyme cocktail-based strategies for plastics upcycling.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 nonzero MHETase concentrations tested. Finally, 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.

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