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Bio-upcycling of polyethylene terephthalate

18 Mar 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: A novel value-chain for PET upcycling is presented, adding technological flexibility to the global challenge of end-of-life management of plastics.
Abstract: Over 359 million tons of plastics were produced worldwide in 2018, with significant growth expected in the near future, resulting in the global challenge of end-of-life management. The recent identification of enzymes that degrade plastics previously considered non-biodegradable opens up opportunities to steer the plastic recycling industry into the realm of biotechnology. Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA. The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel value-chain for PET upcycling, adding technological flexibility to the global challenge of end-of-life management of plastics. Graphical abstract
Topics: Polyethylene terephthalate (56%), Bioplastic (54%), Ethylene glycol (52%), Plastic recycling (52%)

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1
Bio-upcycling of polyethylene terephthalate
Till Tiso
1,§
, Tanja Narancic
2,3,§
, Ren Wei
4,#
, Eric Pollet
5
, Niall Beagan
2
, Katja Schröder
1
, Annett
Honak
4
, Mengying Jiang
5,8
, Shane T. Kenny
6
, Nick Wierckx
1,7
, Rémi Perrin
8
, Luc Avérous
5
,
Wolfgang Zimmermann
4
, Kevin O’Connor
2,3
*, and Lars M. Blank
1
*
1
iAMB - Institute of Applied Microbiology. ABBt - Aachen Biology and Biotechnology, RWTH
Aachen University, Worringerweg 1, D-52074 Aachen, Germany
2
BEACON - SFI Bioeconomy Research centre, University College Dublin, Belfield, Dublin 4,
Ireland
3
School of Biomolecular and Biomedical Science and UCD Earth Institute, University College
Dublin, Belfield, Dublin 4, Ireland
4
Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig
University, Johannisallee 23, D-04103 Leipzig, Germany
5
BioTeam/ICPEES-ECPM, UMR CNRS 7515, Strasbourg University, 25 rue Becquerel, F-
67087 Strasbourg Cedex 2, France
6
Bioplastech Ltd., NovaUCD, Belfield Innovation Park, University College Dublin, Belfield,
Dublin 4, Ireland
7
Institute of Bio- and Geosciences IBG-1: Biotechnology, Forschungszentrum Jülich, 52425
Jülich, Germany
8
SOPREMA, 14 rue de Saint-Nazaire, F-67025 Strasbourg Cedex, France
§
These authors contributed equally to the work
#
Current address for Ren Wei: Department of Biotechnology and Enzyme Catalysis, Institute
of Biochemistry, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

2
*Corresponding authors:
Lars M. Blank
iAMB - Institute of Applied Microbiology. ABBt - Aachen Biology and Biotechnology, RWTH
Aachen University, Worringerweg 1, D-52074 Aachen, Germany
Phone: +49 241 80 26600 (office), +49 241 80 622180 (fax); e-mail: lars.blank@rwth-
aachen.de
Kevin O’Connor
UCD Earth Institute and School of Biomolecular and Biomedical Science, BEACON -
Bioeconomy Research Centre, University College Dublin, Belfield, Dublin 4, Ireland
Phone: +353 1 716 4000, e-mail: kevin.oconnor@ucd.ie
Abbreviations: EG ethylene glycol, TA terephthalic acid terephthalate, PET - polyethylene
terephthalate, PHA polyhydroxyalkanoate, HAA hydroxyalkanoyloxy-alkanoate, MHET
mono-(2-hydroxyethyl)TA
Keywords: polyethylene terephthalate (PET) degradation, metabolic engineering, biopolymers,
polyhydroxyalkanoate (PHA), bio-upcycling, Pseudomonas putida, bioplastic, synthetic biology
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

3
Graphical abstract
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

4
Abstract
Over 359 million tons of plastics were produced worldwide in 2018, with significant growth
expected in the near future, resulting in the global challenge of end-of-life management. The
recent identification of enzymes that degrade plastics previously considered non-biodegradable
opens up opportunities to steer the plastic recycling industry into the realm of biotechnology.
Here, we present the sequential conversion of polyethylene terephthalate (PET) into two types
of bioplastics: a medium chain-length polyhydroxyalkanoate (PHA) and a novel bio-based
poly(amide urethane) (bio-PU). PET films were hydrolyzed by a thermostable polyester
hydrolase yielding 100% terephthalate and ethylene glycol. A terephthalate-degrading
Pseudomonas was evolved to also metabolize ethylene glycol and subsequently produced PHA.
The strain was further modified to secrete hydroxyalkanoyloxy-alkanoates (HAAs), which were
used as monomers for the chemo-catalytic synthesis of bio-PU. In short, we present a novel
value-chain for PET upcycling, adding technological flexibility to the global challenge of end-
of-life management of plastics.
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

5
1 Introduction
One of the challenges humankind faces is the shift to a sustainable plastic industry. In 2018,
359 million tons of plastics have been produced worldwide and this number is growing at a rate
of approximately 3% per annum
1
. Of all the plastic ever produced, only 9% was recycled and
12% was incinerated. The remaining majority is either in use or was landfilled, with a chance
to be released into the environment
2
. Indeed, in 2010 an estimated 5-13 million tons of plastic
ultimately ended up in the ocean
3
. While plastic, due to its lightweight and sturdiness, has many
environmentally beneficial applications, the environmental damage caused by plastic must be
arrested by addressing the end-of-life challenge.
State-of-the art plastic recycling is either via mechanical or chemical methods, or a combination
thereof
4
. An ideal plastic for recycling is polyethylene terephthalate (PET). The main PET
product, beverage bottles, can be specifically collected, avoiding mixed material challenges. In
addition, with its thermoplastic properties such as high melting temperature and the possibility
to process it without the use of additives, PET fulfils many technical recycling criteria. While
in some European countries, PET is collected at quotas above 95%, only approximately 30%
of it is recycled, even under these ideal conditions
5
. Reasons are manifold including cost,
consumer acceptance, and safety regulations surrounding recycled material, to name a few. An
alternative way to increase plastic recycling is to add additional value to the plastic waste, not
aiming for the same material or consumer good (e.g., bottle-to-bottle recycling), but rather
upcycling to chemicals and materials of higher value. This concept has already been
demonstrated using chemical methods as glycolization
6
, alcoholysis
7
, glycolysis
8,9
, and
organocatalysis
10
.
This upcycling can potentially be achieved by using carbon-rich plastic waste streams as a
substrate for biotechnological processes
5
. Here, PET is degraded into its monomers terephthalic
.CC-BY-NC-ND 4.0 International licensemade available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is
The copyright holder for this preprintthis version posted March 18, 2020. ; https://doi.org/10.1101/2020.03.16.993592doi: bioRxiv preprint

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Brandon C. Knott1, Erika Erickson1, Mark D. Allen, Japheth E. Gado1  +16 moreInstitutions (4)
TL;DR: The characterization of the MHETase enzyme and synergy of the two-enzyme PET depolymerization system may inform enzyme cocktail-based strategies for plastics upcycling and will inform future efforts in the biological deconstruction andUpcycling of mixed plastics.
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