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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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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.

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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.

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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

Authors

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.

Corresponding author

Correspondence to Anthony P. Green.

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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
Extended Data Table 2 Characterisation of cryPET substrate depolymerization under optimised conditions

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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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