Polyethylene terephthalate (PET) is one of the most commonly utilized type of plastic, and PET waste management poses a significant environmental challenge. The most common use of PET is in food packaging, especially drink bottles and clothing, but it also makes up a large portion of the kayaks and other small boats used in rivers. As such, it would be an extremely attractive approach to break down these materials into an environmentally-benign form, regardless of their physical condition. On a larger scale, PET makes up 10% of all produced plastic and, despite being recyclable, may only undergo the process a limited number of times. Overall, this project represents an important undertaking for reducing waste on both a global and local scale.

Our team is tackling the critical challenge of plastic pollution, specifically targeting PET materials. The enzymes PETase and MHETase, produced naturally by soil bacterium Ideonella sakaiensis, work synergistically to degrade PET into environmentally benign monomers, terephthalic acid and ethylene glycol. Although there has been much effort toward highly efficient plastic degradation, a systematic approach using protein engineering and strain engineering to improve plastic degradation is still required to achieve practical applications.

The first aim for the project is to achieve surface display of PETase and MHETase separately in Escherichia coli (E. coli) in order to create whole-cell biocatalysts with the ability to break down PET. E. coli has been used as a platform for expression of these enzymes, but these recombinant proteins are mostly expressed intracellularly, necessitating cell lysis and subsequent protein purification. Our goal of displaying the enzymes on the E. coli surface will be significant in eliminating costs, time, and effort associated with purification of the enzymes and can lead to increased overall efficiency compared to free enzymes.

The second aim for the project is to establish a time-efficient platform to evaluate the efficiency of PET-degrading enzymes. Conventional activity assays require long-time incubation with PET films and rely on HPLC analysis to determine the presence of degradation products. This process is overall rather expensive and time-consuming. The second goal will involve the development of one or more quantitative and high-speed kinetic assays to evaluate the activity of the enzymatic system. We envision that developing such assays is imperative to develop a high-throughput screening platform that can be used by scientists to discover and improve PET-degrading enzymes in the future.

Finally, we aim to improve the activity of PETase through directed evolution using error-prone PCR. This goal will act as a proof of concept and illustrate that the platform has the ability to rapidly create and evaluate improvements made to enzymes and the whole system efficiency. We will adapt error-prone PCR to force the enzymatic DNA sequence to undergo random mutagenesis. Our surface display system and the quick kinetic assay to be developed in our previous two goals will be used here to significantly ease the screening for the PETase variant with faster reaction kinetics.

Our team hopes to lessen the prevalence of PET plastic pollution, both throughout the world and in our local environment. PET is an extremely prevalent waste material that can be found nearly anywhere with little effort but is unfortunately especially common within our local environment in Texas, either as post-consumer waste or microplastics. Landfills are only a temporary solution given the inability to reduce waste without contributing large greenhouse gas emissions. Further adding to the problem, only a portion of the material is recycled, whether because it is improperly discarded or its condition renders it incapable of recycling.

What we believe necessary is a method of disposing and eliminating these materials regardless of their condition, which enzymatic degradation presents. Our idea of developing a whole-cell biocatalyst will be ideal for avoiding costly and time-consuming protein purification processes that hamper the potential industrial applications of PETase. Furthermore, we view this project as a platform for the continued improvement of PETase and other plastic degrading enzymes. Surface display of these enzymes can be combined with improvements made by other teams, such as increased activity or higher stability, and evaluated using the assays which we develop. We hope that our project will take a large step toward making complete recycling of PET in industrial settings a reality while also providing an avenue for efficiently testing variations and making improvements to degradation systems in future works.