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Directed evolution of an efficient and thermostable PET depolymerase

Nature Catalysis volume 5, pages 673–681 (2022)Cite this article

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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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Fig. 1: Workflow for the directed evolution of a PET depolymerase.
Fig. 2: Directed evolution of IsPETaseTS afforded a more thermostable and active catalyst.
Fig. 3: Biocatalytic deconstruction of a range of PET-based materials by HotPETase.
Fig. 4: Structural characterization of HotPETase.

Data availability

Coordinates and structure factors have been deposited in the PDB under accession number 7QVH. Data supporting the findings of this study are available within the paper and its Supplementary Information, or are available from the authors upon reasonable request.

References

  1. Geyer, R., Jambeck, J. & Law, K. Production, use, and fate of all plastics ever made. Sci. Adv. 3, e1700782 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Jambeck, J. R. et al. Plastic waste inputs from land into the ocean. Science 347, 768–771 (2015).

    Article  CAS  PubMed  Google Scholar 

  3. Laville, S. & Taylor, M. A million bottles a minute: world’s plastic binge ‘as dangerous as climate change’. The Guardian (28 June 2017).

  4. The New Plastics Economy: Rethinking the Future of Plastics & Catalysing Action (Ellen MacArthur Foundation, 2017); https://www.ellenmacarthurfoundation.org/publications/the-new-plastics-economy-rethinking-the-future-of-plastics-catalysing-action

  5. Burgess, M., Holmes, H., Sharmina, M. & Shaver, M. P. The future of UK plastics recycling: one bin to rule them all. Resour. Conserv. Recycl. 164, 105191 (2021).

    Article  CAS  Google Scholar 

  6. Rahimi, A. & García, J. M. Chemical recycling of waste plastics for new materials production. Nat. Rev. Chem. 1, 0046 (2017).

    Article  CAS  Google Scholar 

  7. Ellis, L. D. et al. Chemical and biological catalysis for plastics recycling and upcycling. Nat. Catal. 4, 539–556 (2021).

    Article  CAS  Google Scholar 

  8. Singh, A. et al. Techno-economic, life-cycle, and socioeconomic impact analysis of enzymatic recycling of poly(ethylene terephthalate). Joule 5, 2479–2503 (2021).

    Article  CAS  Google Scholar 

  9. Jehanno, C. et al. Organocatalysed depolymerisation of PET in a fully sustainable cycle using thermally stable protic ionic salt. Green. Chem. 20, 1205–1212 (2018).

    Article  CAS  Google Scholar 

  10. Karayannidis, G., Chatziavgoustis, A. P. & Achilias, D. Poly(ethylene terephthalate) recycling and recovery of pure terephthalic acid by alkaline hydrolysis. Adv. Polym. Technol. 21, 250–259 (2002).

    Article  CAS  Google Scholar 

  11. Andrady, A. L. Assessment of environmental biodegradation of synthetic polymers. J. Macromol. Sci. C. 34, 25–76 (1994).

    Article  Google Scholar 

  12. Herrero Acero, E. et al. Enzymatic surface hydrolysis of PET: effect of structural diversity on kinetic properties of cutinases from Thermobifida. Macromolecules 44, 4632–4640 (2011).

    Article  CAS  Google Scholar 

  13. Sulaiman, S. et al. Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Appl. Environ. Microbiol. 78, 1556–1562 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kawai, F. et al. A novel Ca2+-activated, thermostabilized polyesterase capable of hydrolyzing polyethylene terephthalate from Saccharomonospora viridis AHK190. Appl. Microbiol. Biotechnol. 98, 10053–10064 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Ribitsch, D. et al. Hydrolysis of polyethylene terephthalate by p-nitrobenzylesterase from Bacillus subtilis. Biotechnol. Prog. 27, 951–960 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Tournier, V. et al. An engineered PET depolymerase to break down and recycle plastic bottles. Nature 580, 216–219 (2020).

    Article  CAS  PubMed  Google Scholar 

  17. Zimmermann, W. Biocatalytic recycling of polyethylene terephthalate plastic. Philos. Trans. R. Soc. A. 378, 20190273 (2020).

    Article  CAS  Google Scholar 

  18. Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).

    Article  CAS  PubMed  Google Scholar 

  19. Austin, H. P. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc. Natl Acad. Sci. USA 115, E4350–E4357 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Han, X. et al. Structural insight into catalytic mechanism of PET hydrolase. Nat. Commun. 8, 2106 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Joo, S. et al. Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nat. Commun. 9, 382 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Ronkvist, Å. M., Xie, W., Lu, W. & Gross, R. A. Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules 42, 5128–5138 (2009).

    Article  CAS  Google Scholar 

  23. Wei, R. & Zimmermann, W. Biocatalysis as a green route for recycling the recalcitrant plastic polyethylene terephthalate. Microb. Biotechnol. 10, 1302–1307 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Son, H. F. et al. Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradation. ACS Catal. 9, 3519–3526 (2019).

    Article  CAS  Google Scholar 

  25. Son, H. F. et al. Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis. Enzym. Microb. Technol. 141, 109656 (2020).

    Article  CAS  Google Scholar 

  26. Cui, Y. et al. Computational redesign of a PETase for plastic biodegradation under ambient condition by the GRAPE strategy. ACS Catal. 11, 1340–1350 (2021).

    Article  CAS  Google Scholar 

  27. Ma, Y. et al. Enhanced poly(ethylene terephthalate) hydrolase activity by protein engineering. Engineering 4, 888–893 (2018).

    Article  CAS  Google Scholar 

  28. Arnold, F. H. Directed evolution: bringing new chemistry to life. Angew. Chem. Int. Ed. 57, 4143–4148 (2018).

    Article  CAS  Google Scholar 

  29. Zeymer, C. & Hilvert, D. Directed evolution of protein catalysts. Annu. Rev. Biochem. 87, 131–157 (2018).

    Article  CAS  PubMed  Google Scholar 

  30. Zhu, B., Wang, D. & Wei, N. Enzyme discovery and engineering for sustainable plastic recycling. Trends Biotechnol. 40, 22–37 (2021).

    Article  PubMed  CAS  Google Scholar 

  31. Zhong-Johnson, E., Voigt, C. & Sinskey, A. An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films. Sci. Rep. 11, 928 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Then, J. et al. A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate. FEBS Open Bio. 6, 425–432 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ügdüler, S. et al. Towards closed-loop recycling of multilayer and coloured PET plastic waste by alkaline hydrolysis. Green. Chem. 22, 5376–5394 (2020).

    Article  Google Scholar 

  34. Erickson, E. et al. Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation. Chem. Sus. Chem. 15, e202101932 (2022).

    Article  CAS  Google Scholar 

  35. Chen, C.-C. et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis. Nat. Catal. 4, 425–430 (2021).

    Article  CAS  Google Scholar 

  36. Huang, P.-S., Boyken, S. E. & Baker, D. The coming of age of de novo protein design. Nature 537, 320–327 (2016).

    Article  CAS  PubMed  Google Scholar 

  37. Dai, L. et al. Enhancing PET hydrolytic enzyme activity by fusion of the cellulose–binding domain of cellobiohydrolase I from Trichoderma reesei. J. Biotechnol. 334, 47–50 (2021).

    Article  CAS  PubMed  Google Scholar 

  38. Knott, B. C. et al. Characterization and engineering of a two-enzyme system for plastics depolymerization. Proc. Natl Acad. Sci. USA 117, 25476–25485 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Sadler, J. C. & Wallace, S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Green. Chem. 23, 4665–4672 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Rorrer, N. A. et al. Combining reclaimed PET with bio-based monomers enables plastics upcycling. Joule 3, 1006–1027 (2019).

    Article  CAS  Google Scholar 

  41. Anderson, J. C. et al. BglBricks: a flexible standard for biological part assembly. J. Biol. Eng. 4, 1 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. Goldenzweig, A. et al. Automated structure-and sequence-based design of proteins for high bacterial expression and stability molecular cell technology automated structure-and sequence-based design of proteins for high bacterial expression and stability. Mol. Cell 63, 337–346 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Reetz, M. T. & Carballeira, J. D. Iterative saturation mutagenesis (ISM) for rapid directed evolution of functional enzymes. Nat. Protoc. 2, 891–903 (2007).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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. The Graphical Abstract, Fig. 1 and Supplementary Fig. 9 were created with BioRender.com.

Author information

Authors and Affiliations

  1. Manchester Institute of Biotechnology, University of Manchester, Manchester, UK

    Elizabeth L. Bell, Ross Smithson, Jake Foster, Florence J. Hardy, Colin Levy & Anthony P. Green

  2. Department of Materials, University of Manchester, Manchester, UK

    Siobhan Kilbride, Saranarayanan Ramachandran, Sarah J. Haigh & Michael P. Shaver

  3. Department of Chemical Engineering and Analytical Science, University of Manchester, Manchester, UK

    Aleksander A. Tedstone & Arthur A. Garforth

  4. Division of Evolution and Genomic Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

    Philip J. R. Day

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  1. Elizabeth L. Bell
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Contributions

A.P.G. and E.L.B. designed and directed the research. E.L.B. carried out molecular biology, enzyme characterization, assay development, directed evolution experiments and interpreted and presented the data. E.L.B., R.S. and J.F. carried out protein production and purification and performed biochemical assays. R.S. carried out protein crystallization. P.J.R.D. discussed interpretations of biochemical assays. S.K. and M.P.S. carried out polymer characterization measurements and analysed and presented the associated data. S.R. and S.J.H. performed microscopy and interpreted the data. A.A.T. and A.A.G. carried out substrate milling. F.J.H. and C.L. interpreted, analysed and presented structural data, and carried out molecular docking studies. A.P.G., E.L.B., M.P.S. and S.K. wrote the manuscript. All authors discussed the results and reviewed the manuscript.

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Correspondence to Anthony P. Green.

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The authors declare no competing interests.

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Nature Catalysis thanks Rey-Ting Guo, Ioannis Pavlidis and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Overview of the IsPETase directed evolution progression.

The crystal structures represent an overview of the evolution, with the IsPETaseTS protein represented as a turquoise ribbon. The catalytic triad and W185 are shown in ball and stick representation coloured by all atoms, with blue and grey carbon atoms, respectively. The three mutations in IsPETaseTS compared to IsPETaseWT are highlighted as yellow spheres, mutations installed in directed evolution rounds 1-2 as light blue spheres, the rationally inserted additional disulphide bridge (DSB) as black spheres and mutations installed in rounds 3–6 as pink spheres. More details on the evolution outcomes can be found in Supplementary Table 1.

Extended Data Fig. 2 Comparison of reactions with IsPETaseTS and HotPETase over a range of temperatures.

48 h time-courses of cryPET reactions, showing the mean total concentration of released MHET and TPA, with HotPETase (yellow-pink) and IsPETaseTS (blues) over time, using 0.4% cryPET substrate loading (4 g L−1) and 0.29 mg g−1 enzyme loading (0.04 μM). Reactions were performed in pH 9.2, 50 mM Gly-OH buffer, 4% BugBuster, in triplicate, with error bars representing the s.d. of the replicate measurements.

Extended Data Fig. 3 Comparison of reactions with HotPETase and LCCICCG over a range of temperatures.

48 h time-courses of cryPET reactions, showing the mean total concentration of released MHET and TPA, with HotPETase (yellows-pinks) and LCCICCG (greens) over time, at a range of temperatures, using 0.4% cryPET substrate loading (4 g L−1) and 0.29 mg g−1 enzyme loading (0.04 μM). LCCICCG was assayed in its reported optimal operating buffer: pH 8, 100 mM K-Pi; IsPETase and its derivatives were assayed under the library screening buffer conditions: pH 9.2, 50 mM Gly-OH buffer, 4% BugBuster. Reactions were carried out in triplicate, with error bars representing the s.d. of the replicate measurements.

Extended Data Fig. 4 Comparison of reactions with additional enzyme or substrate added.

48 h time-courses showing the mean total concentration of released MHET and TPA, where following reaction with HotPETase at 60 °C for 24 h (pink) (using 0.4% cryPET substrate loading (4 g L−1) and 0.29 mg g−1 enzyme loading (0.04 μM)), either 0.29 mg g−1 fresh enzyme (0.04 μM) (blue) or 4 g L−1 fresh cryPET substrate (yellow) was added. Reactions were performed in pH 9.2, 50 mM Gly-OH buffer, 4% BugBuster, in triplicate, with error bars representing the s.d. of the replicate measurements.

Extended Data Fig. 5 Comparison of HotPETase activity with different substrates at 40 °C and 60 °C under library screening conditions and optimised reaction conditions.

Bar chart showing the mean total concentration of released MHET and TPA accumulated over 3 h (light pink) and 24 h (dark pink) at either 40 °C or 60 °C in reactions using HotPETase with different PET substrates (crystalline PET powder (cryPET), milled bottle-grade PET (bgPET), PET/PE composite film lid (PET/PE), 0.4% total substrate loading (4 g L−1)). Reactions were performed using either library hit screening conditions (A): 0.29 mg g−1 enzyme loading (0.04 μM), pH 9.2, 50 mM Gly-OH, 4% BugBuster or optimised conditions (B): 3.62 mg g−1 enzyme loading (0.5 μM), pH 9.7, 50 mM Gly-OH buffer, 4% BugBuster. Reactions were carried out in triplicate; error bars represent the s.d. of the replicate measurements; each replicate measurement is represented as a black circle.

Extended Data Fig. 6 Comparison HotPETase activity under standard and optimised reaction conditions.

24 h time-courses of reactions conducted at 60 °C with HotPETase, showing the mean percentage of cryPET depolymerized (0.4% cryPET substrate loading (4 g L−1)), calculated using the concentration of MHET and TPA produced. Standard conditions were: 0.29 mg g−1 enzyme loading (0.04 μM), library screening buffer: pH 9.2, 50 mM Gly-OH, 4% BugBuster (dashed line). Optimised reaction conditions were: 3.62 mg g−1 enzyme loading (0.5 μM), pH 9.7, 50 mM Gly-OH, 4% BugBuster (solid line). Reactions were carried out in triplicate; error bars represent the s.d. of the replicate measurements.

Extended Data Fig. 7 Comparison of crystal structures and features of HotPETase and IsPETaseTS.

(a) A global superposition of IsPETaseTS (yellow) and HotPETase (light blue). Mutations in IsPETaseTS compared to IsPETaseWT are highlighted with yellow spheres. Mutations installed during directed evolution are highlighted with pink spheres. The rationally inserted disulphide bridge is highlighted with black spheres. The catalytic triad and W185 are in ball and stick representation coloured by all atoms, with blue and grey carbon atoms, respectively. (b) The disulphide bridge is correctly formed between C233 and C282 in HotPETase. Electron density is 2Fo-Fc contoured at 1 sigma (blue) and 2 sigma (yellow). (c) The conversion of P181 in IsPETaseTS to V181 in HotPETase (highlighted pink) results in extension of β-sheet 6, and the formation of an additional hydrogen bond (dashed lines) to L199. (d) In HotPETase, the wobbling tryptophan (W185, grey sticks), forms a π-stacking interaction (dashed line) with the installed Y214 (pink sticks).

Extended Data Fig. 8 Comparison of HotPETase and HotPETaseLR.

(a) Protein melt curves for HotPETase and HotPETase K212N, E213S, Y214S (HotPETaseLR). Melt curve readings were carried out in triplicate. (b) 24 h time-courses, showing the mean total concentration of released MHET and TPA, in reactions at either 40 °C or 65 °C with either HotPETase or HotPETaseLR, using 0.4% cryPET substrate loading (4 g L−1) and 0.29 mg g−1 enzyme loading (0.04 μM). Reactions were performed in pH 9.2, 50 mM Gly-OH buffer, 4% BugBuster, in triplicate; error bars represent the s.d. of the replicate measurements.

Extended Data Table 1 PET substrate characterization before and after enzyme reactions
Full size table
Extended Data Table 2 Characterisation of cryPET substrate depolymerization under optimised conditions
Full size table

Supplementary information

Supplementary Information

Supplementary Methods, Figs. 1–23, Tables 1–4 and references.

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Bell, E.L., Smithson, R., Kilbride, S. et al. Directed evolution of an efficient and thermostable PET depolymerase. Nat Catal 5, 673–681 (2022). https://doi.org/10.1038/s41929-022-00821-3

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