Engineering and Application of a Thermostable MHETase for PET Depolymerization

Murphy, Natasha P. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)] (ORCID:0000000169159990); Gado, Japheth E. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)]; Neumann, Tabea [University of Hamburg (Germany)]; Perez-Garcia, Pablo [University of Hamburg (Germany)]; Komp, Evan [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)]; Avilan, Luisana [University of Portsmouth (United Kingdom)]; Mathews, Irimpan I. [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)] (ORCID:0000000162543519); Bell, Elizabeth L. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:000000028247743X); Norton-Baker, Brenna [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)]; Clark, Matilda [University of Portsmouth (United Kingdom)]; Garcia, Rebecca R. [National Laboratory of the Rockies (NLR), Golden, CO (United States)]; Alt, Hannah M. [National Laboratory of the Rockies (NLR), Golden, CO (United States)]; Sarangi, Ritimukta [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)] (ORCID:0000000227642279); Pickford, Andrew R. [University of Portsmouth (United Kingdom)] (ORCID:0000000272370030); Streit, Wolfgang R. [University of Hamburg (Germany)] (ORCID:0000000176177396); McGeehan, John E. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States)]; Gauthier, Nicholas P. [Harvard Medical School, Boston, MA (United States); Dana-Farber Cancer Institute, Boston, MA (United States)]; Beckham, Gregg T. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)] (ORCID:000000023480212X)

doi:10.1021/acssuschemeng.6c01404

DOE OSTI · 3377299

Engineering and Application of a Thermostable MHETase for PET Depolymerization

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Abstract

Enzymatic hydrolysis of poly(ethylene terephthalate) (PET) releases mono(2-hydroxyethyl) terephthalate (MHET) as a major product, the accumulation of which can prolong reactor residence times and complicate downstream monomer separations. The use of a MHETase enzyme can enable MHET hydrolysis to the monomers, terephthalic acid and ethylene glycol, but industrial PETases typically operate at thermophilic temperatures and the well-known MHETase from Ideonella sakaiensis is a mesophilic enzyme, thus warranting the development of thermophilic MHETases. Here, we characterize thermostable MHET-active enzymes from a natural diversity screen by applying a hidden Markov model based on the previously reported, archaeal ferulic acid esterase, PET46. We identified enzymes with higher thermostability than PET46 and quantified their MHETase activity in reactions at 70 °C. The crystal structure of MHT077, the homologue with the highest MHETase activity and an apparent melting temperature (T m,app ) of 94.6 °C, informed site saturation mutagenesis in the active site and lid-domain interface. MHT077 exhibited a ∼100-fold slower unfolding rate at 65 °C than PET46, indicating substantially greater kinetic stability. In parallel, we applied evolution-informed design, a probabilistic model that leverages coevolutionary patterns in large multiple sequence alignments, to improve the activity and thermostability of five ferulic acid esterases. One design, EV-MHT043–5 was identified with a comparable thermostability (T m,app = 96.1 °C) and a 3-fold improvement in its MHETase activity relative to the wildtype enzyme, MHT043. Combination variants of beneficial mutations were screened and afforded a variant, MHT077 LFK , which reduced MHET accumulation in bioreactor experiments with postconsumer PET waste. Overall, this study expands the known MHET-hydrolyzing protein scaffolds available for enzymatic PET recycling.

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY 42 ENGINEERING Enzymes Genetics High-throughput assay Interfacial biocatalysis Peptides and proteins Polymers Protein engineering Terephthalate Thermal stability Thermodynamic properties enzymes high-throughput assay interfacial biocatalysis polymers protein engineering

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Murphy, Natasha P. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)] (ORCID:0000000169159990), Gado, Japheth E. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)], Neumann, Tabea [University of Hamburg (Germany)], Perez-Garcia, Pablo [University of Hamburg (Germany)], Komp, Evan [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)], Avilan, Luisana [University of Portsmouth (United Kingdom)], Mathews, Irimpan I. [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)] (ORCID:0000000162543519), Bell, Elizabeth L. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States)] (ORCID:000000028247743X), Norton-Baker, Brenna [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)], Clark, Matilda [University of Portsmouth (United Kingdom)], Garcia, Rebecca R. [National Laboratory of the Rockies (NLR), Golden, CO (United States)], Alt, Hannah M. [National Laboratory of the Rockies (NLR), Golden, CO (United States)], Sarangi, Ritimukta [SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)] (ORCID:0000000227642279), Pickford, Andrew R. [University of Portsmouth (United Kingdom)] (ORCID:0000000272370030), Streit, Wolfgang R. [University of Hamburg (Germany)] (ORCID:0000000176177396), McGeehan, John E. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States)], Gauthier, Nicholas P. [Harvard Medical School, Boston, MA (United States); Dana-Farber Cancer Institute, Boston, MA (United States)], Beckham, Gregg T. [National Laboratory of the Rockies (NLR), Golden, CO (United States); BOTTLE Consortium, Golden, CO (United States); USDOE Agile BioFoundry, Emeryville, CA (United States)] (ORCID:000000023480212X). 2026-06-08. Engineering and Application of a Thermostable MHETase for PET Depolymerization. https://doi.org/10.1021/acssuschemeng.6c01404

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