Project Inspiration and Description

In the past years, there have been distressing developments about the scale and impact of plastic pollution. In May, an expedition to Challenger’s Deep, 110,000m below sea level, found traces of plastic waste.[1] 93% of bottled water showed signs of microplastic contamination[2] and traces of microplastics have even been found in human faeces. [3] By 2050, it is suggested there will be more plastic than fish in oceans by weight. [4]

What is PET?

Polyethylene terephthalate (PET) is a polyester, formed by condensation polymerisation of MHET, which itself is formed by esterification of ethylene glycol (EG) and terephthalic acid (TPA). It is a useful and common plastic often used in fibres in clothing, and as food containers.This is due to its chemical resistance, durability, malleability.. However, because of these physical properties, it has become a major contributor to plastic pollution. The degradation rate of PET is significantly slow which make them extremely persistent and hard to dispose. PET bottles require approximately 450 years to be decomposed [5] . Combustion of PET could emit harmful products such as formaldehyde, naphthalene, and styrene. [6] Instead, it takes up space in landfills and oceans. In the latter case, it can harm hundreds of thousands of marine organisms, and bioaccumulate throughout the food chain.

How can PET be bio-degraded efficiently?

Previously, PET degradation mainly performed using cutinase and lipase family enzymes which was not effective due to their low affinity to PET [7] . However, a recent discovery of a plastic-degrading enzyme hints at a biological solution to this important issue.

In 2016, a bacterium named Ideonella Sakaiensis is found in the recycling plants in Japan. It consumes plastic, more specifically, PET. They secrete two enzymes named PETase and MHETase, outside their cells by using appendages. [8] PETase hydrolyses PET into its monomers, MHET but also sometimes, BHET and TPA. Next, MHETase further catalyse the breaking down of MHET into EG and TPA. [8] [9] The bacterium takes up EG as its energy sources to maintain metabolic functions and other life processes such as reproduction and growth while TPA is transported into the cell by TPA transporter proteins and is converted into protocatechuic acid (PCA) by various metabolic reactions. [10][11] PCA is then integrated into other metabolic pathways such as the tricarboxylic acid cycle, and eventually, turned into water and carbon dioxide. [8]

Process of PET degradation by PETase

What makes PETase so efficient?

Three-dimensional structure of IsPETase provides invaluable information for rational design of protein engineering to enhance PET degradation efficiency. For example, substitution of Arg280 at subsite IIc of the substrate binding site into a small hydrophobic residue Ala allow more stable binding of longer substrate, subsequently leading to an increase in PETase activity[7]. Besides, the double mutant S238F/W159H significantly narrow PETase active site, similar to its closest homology T.fusca cutinase exhibits improved PET degradation capacity relative to wild-type PETase[9].These finding opens the possibility of enhancing PETase degradation activity by protein engineering approach.

So, how do we design our mutants?

In our project, we will focus on PET degradation capacity of PETase. For now, its degradation rate is too slow for any feasible industrial use. Therefore, we hope to enhance its function by creating mutants.

When PET enters the substrate binding site of PETase, the benzene rings in the PET have T-stacking interactions with a tryptophan residue next to the cleft while the ester bond is cleaved. This cleavage of ester bonds produces a mixture of MHET, BHET, and TPA. [12]

There are also a few other aromatic residues next to the cleft which have other hydrophobic interactions with the PET. [9][12] By reviewing literature of others attempting site-directed mutagenesis of PETase, we have concluded that increasing the hydrophobic property of the substrate binding cleft can potentially increase the activity of PETase.

We also viewed the structures of other enzymes found to be able to degrade PET. One such enzyme, Thermobifida fusca cutinase, T. fusca cutinase, has a narrower substrate-binding cleft.[7] It also degrades PET relatively more well. Thus, its binding site might be able to accommodate the PET more well. [7]

Combining these hypotheses, we have constructed four mutants of PETase, where we change some residues to make them to be more T. fusca cutinase-like in subunit II of the substrate binding site, and also more hydrophobic. We hope that these mutants perform better at degrading PET than the wild type PETase.

Citations

[1]: Morelle, R. (2019, May 13). Mariana Trench: Deepest-ever sub dive finds plastic bag. BBC News. Retrieved from https://www.bbc.com/news/science-environment-48230157

[2]: Mason, S. A., Welch, V. G., & Neratko, J. (2018). Synthetic Polymer Contamination in Bottled Water. Frontiers in Chemistry, 6. doi:10.3389/fchem.2018.00407Synthetic Polymer Contamination in Bottled Water. (2018). Frontiers in Chemistry, 6. doi:10.3389/fchem.2018.00407

[3]: Wüstneck, B. (2018, October 23). In a first, microplastics found in human poop. Retrieved from https://www.nationalgeographic.com/environment/2018/10/news-plastics-microplastics-human-feces/

[4]: The New Plastics Economy, Rethinking The Future of Plastics (Rep.). (2016). Geneva, Switzerland: The World Economic Forum.

[5]. National Park Service, U.S. Department of the Interior. Time it takes for garbage to decompose in the environment. Available at https://www.des.nh. gov/organization/divisions/water/wmb/coastal/trash/documents/ marine_debris.pdf.

[6]:Sovová, K., Ferus, M., Matulková, I., Španěl, P., Dryahina, K., Dvořák, O., & Civiš, S. (2008). A study of thermal decomposition and combustion products of disposable polyethylene terephthalate (PET) plastic using high resolution fourier transform infrared spectroscopy, selected ion flow tube mass spectrometry and gas chromatography mass spectrometry. Molecular Physics,106(9-10), 1205-1214. doi:10.1080/00268970802077876

[7]: Joo, S., Cho, I. J., Seo, H., Son, H. F., Sagong, H., Shin, T. J., . . . Kim, K. (2018). Structural insight into molecular mechanism of poly(ethylene terephthalate) degradation. Nature Communications, 9(1). doi:10.1038/s41467-018-02881-1

[8]: Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science,351(6278), 1196-1199. doi:10.1126/science.aad6359

[9]: Austin, H. P., Allen, M. D., Donohoe, B. S., Rorrer, N. A., Kearns, F. L., Silveira, R. L., . . . Beckham, G. T. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19). doi:10.1073/pnas.1718804115

[10]: Sasoh, M., Masai, E., Ishibashi, S., Hara, H., Kamimura, N., Miyauchi, K., & Fukuda, M.(2006). Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and Environmental Microbiology, 72(3), 1825-1832. doi:10.1128/aem.72.3.1825-1832.2006

[11]: Hosaka, M., Kamimura, N., Toribami, S., Mori, K., Kasai, D., Fukuda, M., & Masai, E. (2013). Novel Tripartite Aromatic Acid Transporter Essential for Terephthalate Uptake in Comamonas sp. Strain E6. Applied and Environmental Microbiology, 79(19), 6148-6155. doi:10.1128/aem.01600-13

[12]: Han, X., Liu, W., Huang, J., Ma, J., Zheng, Y., Ko, T., . . . Guo, R. (2017). Structural insight into catalytic mechanism of PET hydrolase. Nature Communications, 8(1). doi:10.1038/s41467-017-02255-z