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Background

Polypropylene and its Challenges

Polypropylene (PP) accounts for 14% of global polymer production, yet its recycling rate remains below 1% 1. Approximately 60% of manufactured plastics are ultimately discarded, contributing to landfill accumulation and environmental pollution 2. While most research on plastic biodegradation has targeted hydrolysable polymers like PET, PP remains resistant due to its non-hydrolysable backbone and high crystallinity 3. The chemical structure of PP, consisting of strong covalent bonds [CH3CH6]n, makes it highly resistant to enzymatic attack.

Degradation of PP typically occurs through abiotic pathways, primarily initiated by UV-induced cleavage of C-H bonds. This photochemical process generates hydroperoxides and triggers radical chain reactions, leading to β-scission and the formation of oxygenated fragments like ketones, aldehydes, and olefins. This process can be seen in the figure below.

Figure1A

The Discovery of HIS1

A recent breakthrough identified an enzyme capable of notable PP degradation through hydroxylation. Tan et al. performed a metagenomic screen for bioremediation-related enzyme sequences, leading to the discovery of HIS1, an Fe(II)/2-oxoglutarate-dependent oxygenase originally found in Oryza sativa japonica. This enzyme family employs a conserved 2-His-1-carboxylate facial triad to activate O₂, generating a high-valent Fe(IV)=O intermediate, and abstract hydrogen atoms from otherwise inert C–H bonds before hydroxylation 4.

Figure1B

HIS1 is known to confer resistance to multiple β-triketone herbicides (bTHs), such as benzobicylon (BBC), by catalysing their hydroxylation, which neutralises their HPPD-targeting activity 5. Its demonstrated ability to detoxify a wide range of structurally related substrates highlights its promiscuity and suggests a potential function as a polypropylene oxygenase (PPase) capable of polymer hydroxylation.

Directed Evolution Approaches for PP degradation

Directed evolution (DE) is a powerful strategy for improving biomolecule function. It mimics natural evolution by generating genetic diversity through mutagenesis, followed by screening for desired traits. In this project, we applied a combination of site-directed and combinatorial mutagenesis to target surface-exposed residues of HIS1. The hypothesis was that increasing hydrophobicity at these residues would enhance binding to PP surfaces through stronger hydrophobic interactions.

High-Throughput Screening Assay Development

A major challenge in applying DE to PP degradation is the lack of high-throughput assays capable of detecting subtle enzymatic activity on polymers. To address this, an important goal of this project was to develop a novel screening method that detects functional groups produced by polymer oxidation. This approach would enable the efficient evaluation of the mutant library and provide a valuable tool for other DE projects targeting polymers similar to PP.

  1. NORDAHL, S. L., BARAL, N. R., HELMS, B. A. & SCOWN, C. D. 2023. Complementary roles for mechanical and solvent- based recycling in low- carbon, circular polypropylene. Proceedings of the National Academy of Sciences of the United States of America, 120, 8. DOI: 10.1073/pnas.2306902120 ↩

  2. GEYER, R., JAMBECK, J. R. & LAW, K. L. 2017. Production, use, and fate of all plastics ever made. Science Advances, 3, 5. DOI: 10.1126/sciadv.1700782 ↩

  3. WANG, N., HE, S. B., YANG, B. B., ZHANG, H., LIU, D. D., SONG, P. F., CHEN, T. T., WANG, W. Q., GE, H. H. & MA, J. M. 2024. Crystal structure of HPPD inhibitor sensitive protein from Oryza sativa. Biochemical and Biophysical Research Communications, 704, 5. DOI: 10.1016/j.bbrc.2024.149672 ↩

  4. MARTINEZ, S. & HAUSINGER, R. P. 2015. Catalytic Mechanisms of Fe(II)-and 2-Oxoglutarate-dependent Oxygenases. Journal of Biological Chemistry, 290, 20702-20711. DOI: 10.1074/jbc.R115.648691 ↩

  5. Maeda, H., Murata, K., Sakuma, N., Takei, S., Yamazaki, A., Karim, M. R., Kawata, M., Hirose, S., Kawagishi-Kobayashi, M., Taniguchi, Y., Suzuki, S., Sekino, K., Ohshima, M., Kato, H., Yoshida, H., & Tozawa, Y. (2019). A rice gene that confers broad-spectrum resistance to β-triketone herbicides. Science. https://doi.org/aax0379 ↩