Escherichia coli with surface display of PETase and periplasmic localization of mhetase for enhanced terephthalic acid production from PET

Abstract

Enzymatic degradation of polyethylene terephthalate (PET) has garnered attention as a new PET decomposition technology because of its progression under mild conditions. However, the practical application of these enzymes is significantly hindered by their high purification costs. To address this, we developed an Escherichia coli strain with surface-engineered capabilities for efficient PET degradation by expressing PET-degrading enzymes. Several components were considered and optimized for the efficient degradation of PET using this microorganism catalyst. When comparing the expression systems, cells displaying PETase on the surface using the arabinose-inducible system showed a 2.9-fold improvement in PET degradation compared to those using the isopropyl β-d-1-thiogalactopyranoside-inducible system. Furthermore, cells with both surface-displayed FAST-PETase and localized MHETase in the periplasm exhibited a 6.6-fold increase in PET degradation efficiency compared to the initial pETDuet system. Notably, MHETase expression shifted the product profile toward predominantly terephthalic acid (TPA) formation. These results demonstrate that incorporating MHETase into a PET degradation system predominantly yields TPA, suggesting potential applications for efficient PET upcycling into valuable chemicals.

Keywords

Escherichia coli / Cell surface display / Polyethylene terephthalate / PETase / MHETase / Terephthalic acid

References

[1] Leng Z, Padhan RK, Sreeram A. Production of a sustainable paving material through chemical recycling of waste PET into crumb rubber modified asphalt. J Clean Prod, 2018, 180: 682-8

[2] Zhang J, Wang L, Kannan K. Polyethylene terephthalate and polycarbonate microplastics in pet food and feces from the united States. Environ Sci Technol, 2019, 53(20): 12035-42

[3] Yao Z, Seong HJ, Jang Y-S. Environmental toxicity and decomposition of polyethylene. Ecotoxicol Environ Saf, 2022, 242: 113933

[4] Conroy S, Zhang X. Theoretical insights into chemical recycling of polyethylene terephthalate (PET). Polym Degrad Stab, 2024, 223: 110729

[5] Fiorillo C, Trossaert L, Bezeraj E, Debrie S, Ohnmacht H, Steenberge PHMV, et al.. Molecular and material property variations during the ideal degradation and mechanical recycling of PET. RSC Sustain, 2024, 12: 3596-637

[6] Geyer B, Lorenz G, Kandelbauer A. Recycling of poly(ethylene terephthalate)-A review focusing on chemical methods. eXPRESS Polym Lett, 2016, 10: 559-86

[7] Geyer B, Rohner S, Lorenz G, Kandelbauer A. Designing oligomeric ethylene terephtalate Building blocks by chemical recycling of polyethylene terephtalate. J Appl Polym Sci, 2014, 131(2): 39786

[8] Ronkvist AM, Xie W, Lu W, Gross RA. Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules, 2009, 42(145128-38

[9] Sulaiman S, Yamato S, Kanaya E, Kim JJ, Koga Y, Takano K, 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, 2012, 78(5): 1556-62

[10] Muller RJ, Schrader H, Profe J, Dresler K, Deckwer WD. Enzymatic degradation of poly(ethylene terephthalate): rapid hydrolyse using a hydrolase from T. fusca. Macromol Rapid Commun, 2005, 26(17): 1400-5

[11] Carletti A, Bhattacharya S, Pedroni S, Berto M, Bonettini R, Castagna R, et al.. Functional and structural characterization of PETase SM14 from Marine-Sponge Streptomyces sp. Active on polyethylene terephthalate. ACS Sustain Chem Eng, 2025, 13(20): 7460-8

[12] Yoshida S, Hiraga K, Takehana T, Taniguchi I, Yamaji H, Maeda Y, et al.. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 2016, 351(6278): 1196-9

[13] Son HF, Cho IJ, Joo S, Seo H, Sagong H, Choi SY, et al.. Rational protein engineering of thermo-stable PETase from Ideonella sakaiensis for highly efficient PET degradation. ACS Catal, 2019, 9(4): 3519-26

[14] Cui Y, Chen Y, Liu X, Dong S, Tian Y, Qiao Y, et al.. Computational redesign of a PETase for plastic biodegradation under ambient condition by the GRAPE strategy. ACS Catal, 2021, 11(3): 1340-50

[15] Lu H, Diaz DJ, Czarnecki NJ, Zhu C, Kim W, Shroff R, et al.. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 2022, 604: 662-7

[16] Thomsen TB, Hunt CJ, Meyer AS. Influence of substrate crystallinity and glass transition temperature on enzymatic degradation of polyethylene terephthalate (PET). New Biotechnol, 2022, 69: 28-35

[17] Chen Z, Duan R, Xiao Y, Wei Y, Zhang H, Sun X, et al.. Biodegradation of highly crystallized poly(ethylene terephthalate) through cell surface codisplay of bacterial PETase and Hydrophobin. Nat Commun, 2022, 13: 7138

[18] Huang X, Cao L, Qin Z, Li S, Kong W, Liu Y. Tat-Independent secretion of polyethylene terephthalate hydrolase PETase in Bacillus subtilis 168 mediated by its native signal peptide. New Biotechnol, 2018, 66: 13217-27

[19] Oliveria MVD, Calandrini G, Costa CHS, Souza CGS, Alves CN, Silva JRA, et al.. Evaluating cutinase from Fusarium oxysporum as a biocatalyst for the degradation of nine synthetic polymer. Sci Rep, 2025, 15: 2887

[20] Roussel A, Amara S, Nyyssola A, Mateos-Diaz E, Blangy S, Kontkanen H. A cutinase from Trichoderma Reesei with a Lid-Covered active site and kinetic properties of true lipases. J Mol Biol, 2014, 426: 3757-72

[21] Kim JW, Park SB, Tran QG, Cho DH, Choi DY, Lee YJ, et al.. Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae. New Biotechnol, 2020, 19: 97

[22] Tufvesson P, Lima-Ramos J, Nordblad M, Woodley JM. Guidelines and cost analysis for catalyst production in biocatalytic processes. Org Process Res Dev, 2011, 15(1): 266-74

[23] Heinisch T, Schwizer F, Garabedian B, Csibra E, Jeschek M, Vallapurackal J, et al.. E. coli surface display of Streptavidin for directed evolution of an allylic Deallylase. Chem Sci, 2018, 9: 5383-8

[24] Quehl P, Hollender J, Schuurmann J, Brossette T, Mass R, Jose J. 2016 Co-expression of active human cytochrome P450 1A2 and cytochrome P450 reductase on the cell surface of Escherichia coli. Microb Cell Fact. https://doi.org/10.1186/s12934-016-0427-5.

[25] Bloois EV, Winter RT, Kolmar H, Fraaije MW. Decorating microbes: surface display of proteins on Escherichia coli. Trends Biotechnol, 2011, 29: 79-86

[26] Jose J, Maas RM, Teese MG. Autodisplay of enzymes - Molecular basis and perspectives. J Biotechnol, 2012, 161: 92-103

[27] Yamashita T, Matsumoto T, Yamada R, Ogino H. Display of PETase on the cell surface of Escherichia coli using the anchor protein PgsA. Appl Biochem Biotechnol, 2024, 196: 5471-83

[28] Pinto AV, Ferreira P, Neves RPP, Fernandes PA, Ramos MJ, Magalhaes AL. Reaction mechanism of MHETase, a PET degrading enzyme. ACS Catal, 2021, 11(1610416-28

[29] Narita J, Okano K, Tateno T, Tanino T, Sewaki T, Sung M, et al.. Display of active enzymes on the cell surface of Escherichia coli using PgsA anchor protein and their application to bioconversion. Appl Microbiol Biotechnol, 2006, 70: 564-72

[30] Fohler L, Leibetseder L, Cserjan-Puschmann M, Striendner G. 2024 Manufacturing of the highly active thermophile PETases PHL7 and PHL7mut3 using Escherichia coli. Microb Cell Fact. https://doi.org/10.1186/s12934-024-02551-6.

[31] Nakamura M, Akada R. Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system. J Biosci Bioeng, 2024, 136(6): 548-56

[32] Oda K, Wlodawer A. Development of enzyme-based approaches for recycling PET on an industrial scale. Biochemistry, 2024, 63(4): 369-401

[33] Han W, Zhang J, Chen Q, Xie Y, Zhang M, Qu J, et al.. Biodegradation of poly(ethylene terephthalate) through PETase surface-display: from function to structure. J Hazard Mater, 2024, 461: 132632

[34] Hu J, Chen Y. Constructing Escherichia coli co-display systems for biodegradation of polyethylene terephthalate. Bioresources Bioprocess, 2023, 10: 91

Funding

Fuji Seal Foundation(Fuji Seal Foundation)

RIGHTS & PERMISSIONS

Jiangnan University