Skip to main content
SpringerLink
Log in

Display of PETase on the Cell Surface of Escherichia coli Using the Anchor Protein PgsA

  • Original Article
  • Published:
Applied Biochemistry and Biotechnology Aims and scope Submit manuscript

Abstract

Enzymatic degradation of polyethylene terephthalate (PET) is attracting attention as a new technology because of its mild reaction conditions. However, the cost of purified enzymes is a major challenge for the practical application of this technology. In this study, we attempted to display the surface of the PET-degrading enzyme, PETase, onto Escherichia coli using the membrane anchor, PgsA, from Bacillus subtilis to omit the need for purification of the enzyme. Immunofluorescence staining confirmed that PETase was successfully displayed on the surface of E. coli cells when a fusion of PgsA and PETase was expressed. The surface-displaying E. coli was able to degrade 94.6% of 1 mM bis(2-hydroxyethyl) terephthalate in 60 min, and the PET films were also degraded in trace amounts. These results indicate that PgsA can be used to present active PETase on the cell surface of E. coli. This technique is expected to be applied for efficient PET degradation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Availability of Data and Materials

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  1. Yan, L., Hou, L., Sun, S., & Wu, P. (2020). Dynamic diffusion of disperse dye in a polyethylene terephthalate film from an infrared spectroscopic perspective. Industrial and Engineering Chemistry Research, 59, 7398–7404. https://doi.org/10.1021/acs.iecr.9b07110

    Article  CAS  Google Scholar 

  2. Leng, Z., Padhan, R. K., & Sreeram, A. (2018). Production of a sustainable paving material through chemical recycling of waste PET into crumb rubber modified asphalt. Journal of Cleaner Production, 180, 682–688. https://doi.org/10.1016/j.jclepro.2018.01.171

    Article  CAS  Google Scholar 

  3. Zhang, J., Wang, L., & Kannan, K. (2019). Polyethylene terephthalate and polycarbonate microplastics in pet food and feces from the United States. Environmental Science & Technology, 53, 12035–12042. https://doi.org/10.1021/acs.est.9b03912

    Article  CAS  Google Scholar 

  4. Lebreton, L. C. M., Zwet, J. V. D., Damsteeg, J. W., Slat, B., Andrady, A., & Reisser, J. (2017). River plastic emissions to the world’s oceans. Nature Communications, 8, 15611. https://doi.org/10.1038/ncomms15611

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Shamsaei, M., Aghayan, I., & Kazemi, K. A. (2017). Experimentalinvestigation of using cross-linked polyethylene waste as aggregate in roller compacted concrete pavement. Journal of Cleaner Production, 165, 290–297. https://doi.org/10.1016/j.jclepro.2017.07.109

    Article  CAS  Google Scholar 

  6. Lopez-Fonseca, R., Duque-Ingunza, I., Rivas, B. D., Arnaiz, S., & Gutierrez-Ortiz, J. I. (2010). Chemical recycling of post-consumer PET wastes by glycolysis in the presence of metal salts. Polymer Degradation and Stability, 95, 1022–1028. https://doi.org/10.1016/j.polymdegradstab.2010.03.007

    Article  CAS  Google Scholar 

  7. Badia, J. D., Vilaplana, F., Karlsson, S., & Ribes-Greus, A. (2009). Thermal analysis as a quality tool for assessing the influence of thermo-mechanical degradation on recycled poly (ethylene terephthalate). Polymer Testing, 28, 169–175. https://doi.org/10.1016/j.polymertesting.2008.11.010

    Article  CAS  Google Scholar 

  8. Bartolome, L., Imran, M., Cho, B. G., Al-Masry, W. A., & Kim, D. H. (2012). Recent developments in the chemical recycling of PET. Material Recycling-Trends and Perspectives. https://doi.org/10.5772/33800

    Article  Google Scholar 

  9. Geyer, B., Lorenz, G., & Kandelbauer, A. (2016). Recycling of poly(ethylene terephthalate)-A review focusing on chemical methods. Express Polymer Letters, 10, 559–586. https://doi.org/10.3144/expresspolymlett.2016.53

    Article  CAS  Google Scholar 

  10. Geyer, B., Rohner, S., Lorenz, G., & Kandelbauer, A. (2014). Designing oligomeric ethylene terephtalate building blocks by chemical recycling of polyethylene terephtalate. Journal of Applied Polymer Science, 131, 39786–39786. https://doi.org/10.1002/app.39786

    Article  CAS  Google Scholar 

  11. Kamber, N. E., Tsujii, Y., Keets, K., Waymouth, R. M., Pratt, R. C., Nyce, G. W., & Hedrick, J. L. (2010). The depolymerization of poly (ethylene terephthalate)(PET) using N-heterocyclic carbenes from ionic liquids. Journal of Chemical Education, 87, 519–521. https://doi.org/10.1021/ed800152c

    Article  CAS  Google Scholar 

  12. Ronkvist, A. M., Xie, W., Lu, W., & Gross, R. A. (2009). Cutinase-catalyzed hydrolysis of poly(ethylene terephthalate). Macromolecules, 42, 5128–5138. https://doi.org/10.1021/ma9005318

    Article  CAS  Google Scholar 

  13. Sulaiman, S., Yamato, S., Kanaya, E., Kim, J. J., Koga, Y., Takano, K., & Kanaya, S. (2012). Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach. Applied and Environment Microbiology, 78, 1556–1562. https://doi.org/10.1128/AEM.06725-11

    Article  CAS  Google Scholar 

  14. Muller, R. J., Schrader, H., Profe, J., Dresler, K., & Deckwer, W. D. (2005). Enzymatic degradation of poly(ethylene terephthalate): Rapid hydrolyse using a hydrolase from T. fusca. Macromolecular Rapid Communications, 26, 1400–1405. https://doi.org/10.1002/marc.200500410

    Article  CAS  Google Scholar 

  15. Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K., Miyamoto, K., Kimura, Y., & Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351, 1196–1199. https://doi.org/10.1126/science.aad6359

    Article  PubMed  CAS  Google Scholar 

  16. Ma, Y., Yao, M., Li, B., Ding, M., He, B., Chen, S., Zhou, X., & Yuan, Y. (2018). Enhanced poly (ethylene terephthalate) hydrolase activity by protein engineering. Engneering, 4, 888–893. https://doi.org/10.1016/j.eng.2018.09.007

    Article  CAS  Google Scholar 

  17. Pirillo, V., Orlando, M., Tessaro, D., Pollegioni, L., & Molla, G. (2021). An efficient protein evolution workflow for the improvement of bacterial PET hydrolyzing enzymes. International Journal of Molecular Sciences, 23, 264. https://doi.org/10.3390/ijms23010264

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Cui, Y., Chen, Y., Liu, X., Dong, S., Tian, Y., Qiao, Y., Mitra, R., Han, J., Li, C., Han, X., Liu, W., Chen, Q., Wei, W., Wang, X., Du, W., Tang, S., Xiang, H., Liu, H., Liang, Y., … Wu, B. (2021). Computational redesign of a PETase for plastic biodegradation under ambient condition by the GRAPE strategy. ACS Catalysis, 11, 1340–1350. https://doi.org/10.1021/acscatal.0c05126

    Article  CAS  Google Scholar 

  19. Lu, H., Diaz, D. J., Czarnecki, N. J., Zhu, C., Kim, W., Shroff, R., Acosta, D. J., Alexander, B. R., Cole, H. O., Zhang, Y., Lynd, N. A., Ellington, A. D., & Alper, H. S. (2022). Machine learning-aided engineering of hydrolases for PET depolymerization. Nature, 604, 662–667. https://doi.org/10.1038/s41586-022-04599-z

    Article  PubMed  CAS  Google Scholar 

  20. Tufvesson, P., Lima-Ramos, J., Nordblad, M., & Woodley, J. M. (2011). Guidelines and cost analysis for catalyst production in biocatalytic processes. Organic Process Research & Development, 15, 266–274. https://doi.org/10.1021/op1002165

    Article  CAS  Google Scholar 

  21. Heinisch, T., Schwizer, F., Garabedian, B., Csibra, E., Jeschek, M., Vallapurackal, J., Vallapurackal, J., Pinheiro, V. B., Marliere, P., Panke, S., & Ward, T. R. (2018). E. coli surface display of streptavidin for directed evolution of an allylic deallylase. Chemical Science, 9, 5383–5388. https://doi.org/10.1039/c8sc00484f

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. 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. Microbial Cell Factories, 15. https://doi.org/10.1186/s12934-016-0427-5

  23. Bloois, E. V., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: Surface display of proteins on Escherichia coli. Trends in Biotechnology, 29, 79–86. https://doi.org/10.1016/j.tibtech.2010.11.003

    Article  PubMed  CAS  Google Scholar 

  24. Jose, J., Maas, R. M., & Teese, M. G. (2012). Autodisplay of enzymes-Molecular basis and perspectives. Journal of Biotechnology, 161, 92–103. https://doi.org/10.1016/j.jbiotec.2012.04.001

    Article  PubMed  CAS  Google Scholar 

  25. Verhoeven, G. S., Alexeeva, S., Dogterom, M., & Blaauwen, T. (2009). Differential bacterial surface display of peptides by the transmembrane domain of ompA. PLoS One, 4, e6739. https://doi.org/10.1371/journal.pone.0006739

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. Samuelson, P., Gunneriusson, E., Nygren, P. A., & Stahl, S. (2002). Display of proteins on bacteria. Journal of Biotechnology, 96, 129–154. https://doi.org/10.1016/S0168-1656(02)00043-3

    Article  PubMed  CAS  Google Scholar 

  27. Jose, J., & Meyer, T. F. (2007). The autodisplay story, from discovery to biotechnical and biomedical applications. Microbiology and Molecular Biology Reviews, 71, 600–619. https://doi.org/10.1128/MMBR.00011-07

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Ashiuchi, M., Nawa, C., Kamei, T., Song, J. J., Hong, S. P., Sung, M. H., Soda, K., Yagi, T., & Misono, H. (2001). Physiological and biochemical characteristics of poly γ-glutamate synthetase complex of Bacillus subtilis. European Journal of Biochemistry, 268, 5321–5328. https://doi.org/10.1046/j.0014-2956.2001.02475.x

    Article  PubMed  CAS  Google Scholar 

  29. Narita, J., Okano, K., Tateno, T., Tanino, T., Sewaki, T., Sung, M., Fukuda, H., & Kondo, A. (2006). Display of active enzymes on the cell surface of Escherichia coli using pgsA anchor protein and their application to bioconversion. Applied Microbiology and Biotechnology, 70, 564–572. https://doi.org/10.1007/s00253-005-0111-x

    Article  PubMed  CAS  Google Scholar 

  30. Gallus, S., Peschke, T., Paulsen, M., Burgahn, T., Niemeyer, C. M., & Rabe, K. S. (2020). Surface display of complex enzymes by in situ spycatcher-spytag interaction. ChemBioChem, 21, 2126–2131. https://doi.org/10.1002/cbic.202000102

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Gallus, S., Mittmann, E., & Rabe, K. S. (2022). A modular system for the rapid comparison of different membrane anchors for surface display on Escherichia coli. ChemBioChem, 23, e202100472. https://doi.org/10.1002/cbic.202100472

    Article  PubMed  CAS  Google Scholar 

  32. Ko, M. K., Kim, M. J., Yi, J., Kang, J., Bae, J. H., Sohn, J. H., & Sung, B. H. (2021). A novel protein fusion partner, carbohydrate-binding module family 66, to enhance heterologous protein expression in Escherichia coli. Microbial Cell Factories, 20, 232. https://doi.org/10.1186/s12934-021-01725-w

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Nascimento, B. M., & Nair, N. U. (2020). Characterization of a membrane enzymatic complex for heterologous production of poly-γ-glutamate in E. coli. Metabolic Engineering Communications, 11, e00144. https://doi.org/10.1016/j.mec.2020.e00144

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank Editage (http://www.editage.com) for English language editing.

Funding

This study was supported by a Grant from the Fuji Seal Foundation.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, Osaka Metropolitan University, 1-1 Gakuen-Cho, Naka-Ku, Sakai, Osaka, 599-8531, Japan

    Takuma Yamashita, Takuya Matsumoto, Ryosuke Yamada & Hiroyasu Ogino

Authors
  1. Takuma Yamashita
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Takuya Matsumoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryosuke Yamada
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroyasu Ogino
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by TY and TM. The first draft of the manuscript was written by TY and TM commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Takuya Matsumoto.

Ethics declarations

Ethical Approval

This study was approved by Osaka Metropolitan University and carried out according to the guidelines of the committee at Osaka Metropolitan University.

Consent to Participate

All authors have their consent to participate.

Consent for Publication

All authors have their consent to publish their work.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Key Points

• PETase was successfully displayed on the E. coli surface via PgsA anchor.

• Genetic fusion between PETase and PgsA exhibited its highest activity.

• Displayed PETase efficiently degraded BHET rather than using the crude enzyme.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 203 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yamashita, T., Matsumoto, T., Yamada, R. et al. Display of PETase on the Cell Surface of Escherichia coli Using the Anchor Protein PgsA. Appl Biochem Biotechnol (2024). https://doi.org/10.1007/s12010-023-04837-8

Download citation

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12010-023-04837-8

Keywords

Search

Navigation