Ancestral Sequence Reconstruction Identifies Structural Changes Underlying the Evolution of Ideonella sakaiensis PETase and Variants with Improved Stability and Activity
- Yvonne JohoYvonne JohoManufacturing, Commonwealth Scientific and Industrial Research Organisation, Clayton, Victoria 3168, AustraliaResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Yvonne Joho
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- Vanessa VongsouthiVanessa VongsouthiResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Vanessa Vongsouthi
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- Matthew A. SpenceMatthew A. SpenceResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Matthew A. Spence
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- Jennifer TonJennifer TonResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Jennifer Ton
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- Chloe GomezChloe GomezResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Chloe Gomez
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- Li Lynn TanLi Lynn TanResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Li Lynn Tan
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- Joe A. KaczmarskiJoe A. KaczmarskiResearch School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaARC Centre of Excellence for Innovations in Synthetic Biology, Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Joe A. Kaczmarski
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- Alessandro T. CaputoAlessandro T. CaputoManufacturing, Commonwealth Scientific and Industrial Research Organisation, Clayton, Victoria 3168, AustraliaMore by Alessandro T. Caputo
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- Santana RoyanSantana RoyanManufacturing, Commonwealth Scientific and Industrial Research Organisation, Clayton, Victoria 3168, AustraliaMore by Santana Royan
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- Colin J. Jackson*Colin J. Jackson*Email: [email protected]Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaARC Centre of Excellence for Innovations in Synthetic Biology, Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaARC Centre of Excellence for Innovations in Peptide & Protein Science, Research School of Chemistry, Australian National University, Canberra, ACT 2601, AustraliaMore by Colin J. Jackson
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- Albert Ardevol*Albert Ardevol*Email: [email protected]Manufacturing, Commonwealth Scientific and Industrial Research Organisation, Clayton, Victoria 3168, AustraliaCSIRO Synthetic Biology Future Science Platform, GPO Box 1700, Canberra, ACT 2601, AustraliaMore by Albert Ardevol
Abstract
The improved production, recycling, and removal of plastic waste, such as polyethylene terephthalate (PET), are pressing environmental and economic issues for society. Biocatalytic (enzymatic) PET depolymerization is potentially a sustainable, low-energy solution to PET recycling, especially when compared with current disposal methods such as landfills, incineration, or gasification. IsPETase has been extensively studied for its use in PET depolymerization; however, its evolution from cutinases is not fully understood, and most engineering studies have neglected the majority of the available sequence space remote from the active site. In this study, ancestral protein reconstruction (ASR) has been used to trace the evolutionary trajectory from ancient serine hydrolases to IsPETase, while ASR and the related design approach, protein repair one-stop shop, were used to identify enzyme variants with improved activity and stability. Kinetic and structural characterization of these variants reveals new insights into the evolution of PETase activity and the role of second-shell mutations around the active site. Among the designed and reconstructed variants, we identified several with melting points 20 °C higher than that of IsPETase and two variants with significantly higher catalytic activity.
Cited By
This article is cited by 2 publications.
- Alessandro Crnjar, Aransa Griñen, Shina C. L. Kamerlin, César A. Ramírez-Sarmiento. Conformational Selection of a Tryptophan Side Chain Drives the Generalized Increase in Activity of PET Hydrolases through a Ser/Ile Double Mutation. ACS Organic & Inorganic Au 2023, 3 (2) , 109-119. https://doi.org/10.1021/acsorginorgau.2c00054
- Sebastian Weigert, Pablo Perez‐Garcia, Florian J. Gisdon, Andreas Gagsteiger, Kristine Schweinshaut, G. Matthias Ullmann, Jennifer Chow, Wolfgang R. Streit, Birte Höcker. Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes. Protein Science 2022, 31 (12) https://doi.org/10.1002/pro.4500