Directed evolution of an efficient and thermostable PET depolymerase

Elizabeth L. Bell; Ross Smithson; Siobhan Kilbride; Jake Foster; Florence  J. Hardy; Saranarayanan Ramachandran; Aleksander A. Tedstone; Sarah J. Haigh; Arthur A. Garforth; Philip J. R. Day; Colin Levy; Michael P. Shaver; Anthony P. Green

doi:10.1038/s41929-022-00821-3

Directed evolution of an efficient and thermostable PET depolymerase

Elizabeth L. Bell
, Ross Smithson
, Siobhan Kilbride
, Jake Foster
, Florence J. Hardy
, Saranarayanan Ramachandran
, Aleksander A. Tedstone
, Sarah J. Haigh
, Arthur A. Garforth
, Philip J. R. Day
, Colin Levy
, Michael P. Shaver
, Anthony P. Green

Research output: Contribution to journal › Article › peer-review

428 Citations (Scopus)

Abstract

The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, T_m = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.

Original language	English
Pages (from-to)	673–681
Number of pages	9
Journal	Nature Catalysis
Volume	5
DOIs	https://doi.org/10.1038/s41929-022-00821-3
Publication status	Published - 11 Aug 2022
Externally published	Yes

Funding

We acknowledge generous support from the Biotechnology and Biological Sciences Research Council (David Phillips Fellowship grant no. BB/M027023/1 to A.P.G), the European Research Council (ERC Starter grant no. 757991 to A.P.G), the Medical Research Council and Buddi (MRC iCASE PhD funding to E.L.B), the UK Catalysis Hub (funded through grant nos. EP/R026815/1, EP/K014706/2, EP/K014668/1, EP/K014854/1, EP/K014714/1 and EP/M013219/1), the EPSRC (grant no. EP/R031711/1 to S.J.H. and S.R.), the Henry Royce Institute for Advanced Materials (funded through grant nos. EP/R00661X/1, EP/S019367/1, EP/P025021/1 and EP/P025498/1), and the ISCF Smart Sustainable Plastic Packaging fund (grant no. NE/V01045X/1 with H. Holmes, M. Sharmina, A. Adelekan, T. Holmes). We are grateful to Diamond Light Source for time on beamlines i03, i04 and i04-1 under proposals MX12788-50, MX17773-25, MX17773-34 and MX17773-52, and to Manchester SYNBIOCHEM Centre (grant no. BB/M017702/1) and the Future Biomanufacturing Hub (EP/S01778X/1) for access to their facilities.

Keywords

poly(ethylene terephthalate)
plastic recycling
plastic degrading enzymes

Access to Document

10.1038/s41929-022-00821-3

https://chemrxiv.org/engage/chemrxiv/article-details/61c9db381e13eb8ccc076210Licence: CC BY-NC-ND 4.0

Cite this

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title = "Directed evolution of an efficient and thermostable PET depolymerase",

abstract = "The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, Tm = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.",

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author = "\{L. Bell\}, Elizabeth and Ross Smithson and Siobhan Kilbride and Jake Foster and \{J. Hardy\}, Florence and Saranarayanan Ramachandran and \{A. Tedstone\}, Aleksander and \{J. Haigh\}, Sarah and \{A. Garforth\}, Arthur and \{J. R. Day\}, Philip and Colin Levy and \{P. Shaver\}, Michael and \{P. Green\}, Anthony",

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doi = "10.1038/s41929-022-00821-3",

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AU - L. Bell, Elizabeth

AU - Smithson, Ross

AU - Kilbride, Siobhan

AU - Foster, Jake

AU - J. Hardy, Florence

AU - Ramachandran, Saranarayanan

AU - A. Tedstone, Aleksander

AU - J. Haigh, Sarah

AU - A. Garforth, Arthur

AU - J. R. Day, Philip

AU - Levy, Colin

AU - P. Shaver, Michael

AU - P. Green, Anthony

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N2 - The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, Tm = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.

AB - The recent discovery of IsPETase, a hydrolytic enzyme that can deconstruct poly(ethylene terephthalate) (PET), has sparked great interest in biocatalytic approaches to recycle plastics. Realization of commercial use will require the development of robust engineered enzymes that meet the demands of industrial processes. Although rationally engineered PETases have been described, enzymes that have been experimentally optimized via directed evolution have not previously been reported. Here, we describe an automated, high-throughput directed evolution platform for engineering polymer degrading enzymes. Applying catalytic activity at elevated temperatures as a primary selection pressure, a thermostable IsPETase variant (HotPETase, Tm = 82.5 °C) was engineered that can operate at the glass transition temperature of PET. HotPETase can depolymerize semicrystalline PET more rapidly than previously reported PETases and can selectively deconstruct the PET component of a laminated multimaterial. Structural analysis of HotPETase reveals interesting features that have emerged to improve thermotolerance and catalytic performance. Our study establishes laboratory evolution as a platform for engineering useful plastic degrading enzymes.

KW - poly(ethylene terephthalate)

KW - plastic recycling

KW - plastic degrading enzymes

U2 - 10.1038/s41929-022-00821-3

DO - 10.1038/s41929-022-00821-3

M3 - Article

VL - 5

SP - 673

EP - 681

JO - Nature Catalysis

JF - Nature Catalysis

ER -

Directed evolution of an efficient and thermostable PET depolymerase

Abstract

Funding

Keywords

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