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Title: Characterization and engineering of a plastic-degrading aromatic polyesterase

Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral a/..beta..-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PETmore » replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.« less
Authors:
 [1] ;  [1] ; ORCiD logo [2] ;  [2] ;  [3] ;  [4] ;  [3] ;  [2] ; ORCiD logo [5] ;  [5] ;  [5] ; ORCiD logo [5] ;  [2] ;  [2] ;  [6] ;  [2] ;  [1] ;  [2] ;  [3] ; ORCiD logo [1] more »; ORCiD logo [2] « less
  1. Univ. of Portsmouth (United Kingdom)
  2. National Renewable Energy Lab. (NREL), Golden, CO (United States)
  3. Univ. of South Florida, Tampa, FL (United States)
  4. National Renewable Energy Lab. (NREL), Golden, CO (United States); Science and Technology Facilities Council (STFC), Harwell Campus, Oxford (United Kingdom). Diamond Light Source, Ltd.
  5. Science and Technology Facilities Council (STFC), Harwell Campus, Oxford (United Kingdom). Diamond Light Source, Ltd.
  6. Univ. of Campinas (UNICAMP), Sao Paulo (Brazil)
Publication Date:
Report Number(s):
NREL/JA-5100-70516
Journal ID: ISSN 0027-8424
Grant/Contract Number:
AC36-08GO28308; LDRD
Type:
Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 115; Journal Issue: 19; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), NREL Laboratory Directed Research and Development (LDRD); USDOE
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; biodegradation; poly(ethylene terephthalate); poly(ethylene furanoate); plastics; recycling; cutinase
OSTI Identifier:
1435703
Alternate Identifier(s):
OSTI ID: 1433403

Austin, Harry P., Allen, Mark D., Donohoe, Bryon S., Rorrer, Nicholas A., Kearns, Fiona L., Silveira, Rodrigo L., Pollard, Benjamin C., Dominick, Graham, Duman, Ramona, El Omari, Kamel, Mykhaylyk, Vitaliy, Wagner, Armin, Michener, William E., Amore, Antonella, Skaf, Munir S., Crowley, Michael F., Thorne, Alan W., Johnson, Christopher W., Woodcock, H. Lee, McGeehan, John E., and Beckham, Gregg T.. Characterization and engineering of a plastic-degrading aromatic polyesterase. United States: N. p., Web. doi:10.1073/pnas.1718804115.
Austin, Harry P., Allen, Mark D., Donohoe, Bryon S., Rorrer, Nicholas A., Kearns, Fiona L., Silveira, Rodrigo L., Pollard, Benjamin C., Dominick, Graham, Duman, Ramona, El Omari, Kamel, Mykhaylyk, Vitaliy, Wagner, Armin, Michener, William E., Amore, Antonella, Skaf, Munir S., Crowley, Michael F., Thorne, Alan W., Johnson, Christopher W., Woodcock, H. Lee, McGeehan, John E., & Beckham, Gregg T.. Characterization and engineering of a plastic-degrading aromatic polyesterase. United States. doi:10.1073/pnas.1718804115.
Austin, Harry P., Allen, Mark D., Donohoe, Bryon S., Rorrer, Nicholas A., Kearns, Fiona L., Silveira, Rodrigo L., Pollard, Benjamin C., Dominick, Graham, Duman, Ramona, El Omari, Kamel, Mykhaylyk, Vitaliy, Wagner, Armin, Michener, William E., Amore, Antonella, Skaf, Munir S., Crowley, Michael F., Thorne, Alan W., Johnson, Christopher W., Woodcock, H. Lee, McGeehan, John E., and Beckham, Gregg T.. 2018. "Characterization and engineering of a plastic-degrading aromatic polyesterase". United States. doi:10.1073/pnas.1718804115.
@article{osti_1435703,
title = {Characterization and engineering of a plastic-degrading aromatic polyesterase},
author = {Austin, Harry P. and Allen, Mark D. and Donohoe, Bryon S. and Rorrer, Nicholas A. and Kearns, Fiona L. and Silveira, Rodrigo L. and Pollard, Benjamin C. and Dominick, Graham and Duman, Ramona and El Omari, Kamel and Mykhaylyk, Vitaliy and Wagner, Armin and Michener, William E. and Amore, Antonella and Skaf, Munir S. and Crowley, Michael F. and Thorne, Alan W. and Johnson, Christopher W. and Woodcock, H. Lee and McGeehan, John E. and Beckham, Gregg T.},
abstractNote = {Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral a/..beta..-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.},
doi = {10.1073/pnas.1718804115},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 19,
volume = 115,
place = {United States},
year = {2018},
month = {4}
}

Works referenced in this record:

In-vacuum long-wavelength macromolecular crystallography
journal, March 2016
  • Wagner, Armin; Duman, Ramona; Henderson, Keith
  • Acta Crystallographica Section D Structural Biology, Vol. 72, Issue 3, p. 430-439
  • DOI: 10.1107/S2059798316001078