skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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

Abstract

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:
; ; ORCiD logo; ; ; ; ; ; ORCiD logo; ; ; ORCiD logo; ; ; ; ; ; ; ; ORCiD logo more »; ORCiD logo « less
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE), NREL Laboratory Directed Research and Development (LDRD)
OSTI Identifier:
1433403
Alternate Identifier(s):
OSTI ID: 1435703
Report Number(s):
NREL/JA-5100-70516
Journal ID: ISSN 0027-8424; /pnas/115/19/E4350.atom
Grant/Contract Number:  
LDRD; AC36-08GO28308
Resource Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 115 Journal Issue: 19; Journal ID: ISSN 0027-8424
Publisher:
Proceedings of the National Academy of Sciences
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; biodegradation; poly(ethylene terephthalate); poly(ethylene furanoate); plastics; recycling; cutinase

Citation Formats

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., 2018. 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. Tue . "Characterization and engineering of a plastic-degrading aromatic polyesterase". United States. doi:10.1073/pnas.1718804115.
@article{osti_1433403,
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}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1073/pnas.1718804115

Citation Metrics:
Cited by: 34 works
Citation information provided by
Web of Science

Save / Share:

Works referenced in this record:

Production, use, and fate of all plastics ever made
journal, July 2017

  • Geyer, Roland; Jambeck, Jenna R.; Law, Kara Lavender
  • Science Advances, Vol. 3, Issue 7
  • DOI: 10.1126/sciadv.1700782

Geometrical reaction coordinates. II. Nucleophilic addition to a carbonyl group
journal, July 1973

  • Burgi, H. B.; Dunitz, J. D.; Shefter, Eli.
  • Journal of the American Chemical Society, Vol. 95, Issue 15
  • DOI: 10.1021/ja00796a058

Identification and Characterization of Bacterial Cutinase
journal, July 2008

  • Chen, Sheng; Tong, Xing; Woodard, Ronald W.
  • Journal of Biological Chemistry, Vol. 283, Issue 38
  • DOI: 10.1074/jbc.M800848200

Structural and functional studies on a thermostable polyethylene terephthalate degrading hydrolase from Thermobifida fusca
journal, April 2014

  • Roth, Christian; Wei, Ren; Oeser, Thorsten
  • Applied Microbiology and Biotechnology, Vol. 98, Issue 18
  • DOI: 10.1007/s00253-014-5672-0

Cutinase-Catalyzed Hydrolysis of Poly(ethylene terephthalate)
journal, July 2009

  • Ronkvist, Åsa M.; Xie, Wenchun; Lu, Wenhua
  • Macromolecules, Vol. 42, Issue 14
  • DOI: 10.1021/ma9005318

Evidence of Polyethylene Biodegradation by Bacterial Strains from the Guts of Plastic-Eating Waxworms
journal, November 2014

  • Yang, Jun; Yang, Yu; Wu, Wei-Min
  • Environmental Science & Technology, Vol. 48, Issue 23
  • DOI: 10.1021/es504038a

Fungal Cellulases
journal, January 2015

  • Payne, Christina M.; Knott, Brandon C.; Mayes, Heather B.
  • Chemical Reviews, Vol. 115, Issue 3
  • DOI: 10.1021/cr500351c

Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate
journal, April 2017


Plastic waste as a novel substrate for industrial biotechnology: Plastic waste as substrate for biotechnology
journal, August 2015

  • Wierckx, Nick; Prieto, M. Auxiliadora; Pomposiello, Pablo
  • Microbial Biotechnology, Vol. 8, Issue 6
  • DOI: 10.1111/1751-7915.12312

Screening of commercial enzymes for poly(ethylene terephthalate) (PET) hydrolysis and synergy studies on different substrate sources
journal, April 2017

  • de Castro, Aline Machado; Carniel, Adriano; Nicomedes Junior, José
  • Journal of Industrial Microbiology & Biotechnology, Vol. 44, Issue 6
  • DOI: 10.1007/s10295-017-1942-z

Microbial enzymes for the recycling of recalcitrant petroleum‐based plastics: how far are we?
journal, March 2017


Polyethylene bio-degradation by caterpillars of the wax moth Galleria mellonella
journal, April 2017

  • Bombelli, Paolo; Howe, Christopher J.; Bertocchini, Federica
  • Current Biology, Vol. 27, Issue 8
  • DOI: 10.1016/j.cub.2017.02.060

Enzymatic Surface Hydrolysis of PET: Effect of Structural Diversity on Kinetic Properties of Cutinases from Thermobifida
journal, June 2011

  • Herrero Acero, Enrique; Ribitsch, Doris; Steinkellner, Georg
  • Macromolecules, Vol. 44, Issue 12
  • DOI: 10.1021/ma200949p

Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET Hydrolysis
journal, May 2013

  • Ribitsch, Doris; Yebra, Antonio Orcal; Zitzenbacher, Sabine
  • Biomacromolecules, Vol. 14, Issue 6
  • DOI: 10.1021/bm400140u

Poly(butylene succinate) and its copolymers: Research, development and industrialization
journal, November 2010


The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies
journal, December 2013

  • Knott, Brandon C.; Haddad Momeni, Majid; Crowley, Michael F.
  • Journal of the American Chemical Society, Vol. 136, Issue 1
  • DOI: 10.1021/ja410291u

Plastic as a Persistent Marine Pollutant
journal, October 2017


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

Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6
journal, March 2006


Bioremediation 3 . 0 : Engineering pollutant-removing bacteria in the times of systemic biology
journal, November 2017


Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: Part 1. Chemical and Physical Characterization and Isotopic Tests
journal, October 2015

  • Yang, Yu; Yang, Jun; Wu, Wei-Min
  • Environmental Science & Technology, Vol. 49, Issue 20
  • DOI: 10.1021/acs.est.5b02661

Green and sustainable manufacture of chemicals from biomass: state of the art
journal, January 2014


Characterization of a new cutinase from Thermobifida alba for PET-surface hydrolysis
journal, December 2011

  • Ribitsch, Doris; Acero, Enrique Herrero; Greimel, Katrin
  • Biocatalysis and Biotransformation, Vol. 30, Issue 1
  • DOI: 10.3109/10242422.2012.644435

Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms: Part 2. Role of Gut Microorganisms
journal, October 2015

  • Yang, Yu; Yang, Jun; Wu, Wei-Min
  • Environmental Science & Technology, Vol. 49, Issue 20
  • DOI: 10.1021/acs.est.5b02663

The role of short-range Cys171–Cys178 disulfide bond in maintaining cutinase active site integrity: A molecular dynamics simulation
journal, December 2009

  • Matak, Mehdi Youssefi; Moghaddam, Majid Erfani
  • Biochemical and Biophysical Research Communications, Vol. 390, Issue 2
  • DOI: 10.1016/j.bbrc.2009.09.073

A bacterium that degrades and assimilates poly(ethylene terephthalate)
journal, March 2016


Polylactic Acid Technology
journal, December 2000


Plastic Accumulation in the North Atlantic Subtropical Gyre
journal, August 2010


Enhanced Cutinase-Catalyzed Hydrolysis of Polyethylene Terephthalate by Covalent Fusion to Hydrophobins
journal, March 2015

  • Ribitsch, Doris; Herrero Acero, Enrique; Przylucka, Agnieszka
  • Applied and Environmental Microbiology, Vol. 81, Issue 11
  • DOI: 10.1128/AEM.04111-14

Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments
journal, March 2013

  • Madhavi Sastry, G.; Adzhigirey, Matvey; Day, Tyler
  • Journal of Computer-Aided Molecular Design, Vol. 27, Issue 3
  • DOI: 10.1007/s10822-013-9644-8

Observations of floating anthropogenic litter in the Barents Sea and Fram Strait, Arctic
journal, October 2015


Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects
journal, January 2006

  • Sherman, Woody; Day, Tyler; Jacobson, Matthew P.
  • Journal of Medicinal Chemistry, Vol. 49, Issue 2
  • DOI: 10.1021/jm050540c

A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry
journal, January 2009

  • Chen, Guo-Qiang
  • Chemical Society Reviews, Vol. 38, Issue 8
  • DOI: 10.1039/b812677c

Stacking and T-shape Competition in Aromatic−Aromatic Amino Acid Interactions
journal, May 2002

  • Chelli, Riccardo; Gervasio, Francesco Luigi; Procacci, Piero
  • Journal of the American Chemical Society, Vol. 124, Issue 21
  • DOI: 10.1021/ja0121639

Enzymatic surface hydrolysis of poly(ethylene furanoate) thin films of various crystallinities
journal, January 2017

  • Weinberger, S.; Haernvall, K.; Scaini, D.
  • Green Chemistry, Vol. 19, Issue 22
  • DOI: 10.1039/C7GC02905E

Bacterial lipases
journal, September 1994


A Review of Plastic Waste Biodegradation
journal, January 2005

  • Zheng, Ying; Yanful, Ernest K.; Bassi, Amarjeet S.
  • Critical Reviews in Biotechnology, Vol. 25, Issue 4
  • DOI: 10.1080/07388550500346359

Release of synthetic microplastic plastic fibres from domestic washing machines: Effects of fabric type and washing conditions
journal, November 2016


Computing the Role of Near Attack Conformations in an Enzyme-Catalyzed Nucleophilic Bimolecular Reaction
journal, December 2014

  • Sadiq, S. Kashif; Coveney, Peter V.
  • Journal of Chemical Theory and Computation, Vol. 11, Issue 1
  • DOI: 10.1021/ct5008845

Organocatalytic depolymerization of poly(ethylene terephthalate)
journal, January 2011

  • Fukushima, Kazuki; Coulembier, Olivier; Lecuyer, Julien M.
  • Journal of Polymer Science Part A: Polymer Chemistry, Vol. 49, Issue 5
  • DOI: 10.1002/pola.24551

Plastic debris in the open ocean
journal, June 2014

  • Cozar, A.; Echevarria, F.; Gonzalez-Gordillo, J. I.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 28
  • DOI: 10.1073/pnas.1314705111

Microbial biotechnology addressing the plastic waste disaster
journal, July 2017


Towards new enzymes for biofuels: lessons from chitinase research
journal, May 2008


Replacing fossil based PET with biobased PEF; process analysis, energy and GHG balance
journal, January 2012

  • Eerhart, A. J. J. E.; Faaij, A. P. C.; Patel, M. K.
  • Energy & Environmental Science, Vol. 5, Issue 4
  • DOI: 10.1039/c2ee02480b

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica
journal, May 2017

  • Gamerith, Caroline; Vastano, Marco; Ghorbanpour, Sahar M.
  • Frontiers in Microbiology, Vol. 8
  • DOI: 10.3389/fmicb.2017.00938

The α / β hydrolase fold
journal, January 1992

  • Ollis, David L.; Cheah, Eong; Cygler, Miroslaw
  • "Protein Engineering, Design and Selection", Vol. 5, Issue 3
  • DOI: 10.1093/protein/5.3.197

Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation
journal, January 2018


Synthesis and degradability of copolyesters of 2, 5-furandicarboxylic acid, lactic acid, and ethylene glycol
journal, November 2015


Microbial degradation and deterioration of polyethylene – A review
journal, March 2014

  • Restrepo-Flórez, Juan-Manuel; Bassi, Amarjeet; Thompson, Michael R.
  • International Biodeterioration & Biodegradation, Vol. 88
  • DOI: 10.1016/j.ibiod.2013.12.014

Structural insight into catalytic mechanism of PET hydrolase
journal, December 2017


Enzymatic Degradation of Poly(ethylene terephthalate): Rapid Hydrolyse using a Hydrolase fromT. fusca
journal, September 2005

  • Müller, Rolf-Joachim; Schrader, Hedwig; Profe, Jörn
  • Macromolecular Rapid Communications, Vol. 26, Issue 17
  • DOI: 10.1002/marc.200500410

Enzymatic surface modification of poly(ethylene terephthalate)
journal, December 2005


Passport Initiative fosters applied science
journal, August 2016


Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate-Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach
journal, December 2011

  • Sulaiman, Sintawee; Yamato, Saya; Kanaya, Eiko
  • Applied and Environmental Microbiology, Vol. 78, Issue 5
  • DOI: 10.1128/AEM.06725-11

Plastic waste associated with disease on coral reefs
journal, January 2018


Chemical Recycling of Poly(ethylene terephthalate)
journal, April 1997

  • Paszun, Daniel; Spychaj, Tadeusz
  • Industrial & Engineering Chemistry Research, Vol. 36, Issue 4
  • DOI: 10.1021/ie960563c

Plastic waste inputs from land into the ocean
journal, February 2015


Tailoring cutinase activity towards polyethylene terephthalate and polyamide 6,6 fibers
journal, March 2007