Characterization and engineering of a two-enzyme system for plastics depolymerization
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- Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401,
- Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom,
- Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401,, Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506,
- Department of Chemistry, University of South Florida, Tampa, FL 33620,
- Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401,, Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717,
- Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717,
- Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506,
- Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401
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 sakaiensiswas reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, theI. sakaiensisPETase 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.
- Research Organization:
- National Renewable Energy Lab. (NREL), Golden, CO (United States)
- Sponsoring Organization:
- USDOE Office of Energy Efficiency and Renewable Energy (EERE), Energy Efficiency Office. Advanced Manufacturing Office; USDOE Office of Energy Efficiency and Renewable Energy (EERE), Energy Efficiency Office. Building Technologies Office; USDOE Laboratory Directed Research and Development (LDRD) Program; Research England; Biotechnology and Biological Sciences Research Council (BBSRC); National Science Foundation (NSF); National Institutes of Health (NIH)
- Grant/Contract Number:
- AC36-08GO28308; SC0011297TDD; BB/P011918/1; 3900101301; SC0011297; 1R01GM129519-01; CHE-1464946; CBET-1552355; MCB-1714556
- OSTI ID:
- 1668418
- Alternate ID(s):
- OSTI ID: 1772992
- Report Number(s):
- NREL/JA-2A00-76584
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 117 Journal Issue: 41; ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
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