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Ultrahigh-throughput protein evolution for polyethylene biodegradation

Periodic Reporting for period 1 - UTPE PEB (Ultrahigh-throughput protein evolution for polyethylene biodegradation)

Reporting period: 2020-10-01 to 2022-09-30

Polyethylene (PE) is the most abundantly produced plastic in the world, accounting for 30-40% of all synthetic polymers, with an annual global production of nearly 100 million tonnes in 2018. It is found in an extensive array of commonly used items, such as grocery bags, milk cartons and sponges. Due to its low economic cost, it is frequently adopted for single use functions, resulting in large quantities of PE waste. PE is durable, which is appealing for commercial purposes, but creates a major environmental problem as it can indefinitely persist in landfills after being discarded, with only around 10% of the total PE mass produced being recycled. PE degradation (both abiotic and biotic) occurs at a rate that cannot keep pace with current production, inevitably resulting in a build-up of plastic in the environment with detrimental consequences.
The biodegradability of PE could be increased by enhancing natural biological processes through directed protein evolution. It has been known for nearly 50 years that degradation of PE is affected by microbes. Many studies have identified organisms capable of facilitating the degradation of PE, and in some cases the proteins responsible. However, these strains and enzymes are typically poorly characterized or inefficient. Directed protein can be used to improve the efficiency of enzymes, sometimes resulting in increases of reaction rates by many orders of magnitude. However, this impactful technology has yet to be applied to the challenge of polymer biodegradation. We have isolated bacterial strains capable of growth on Polyethylene as a sole carbon source, and have identified a protein element involved. This protein element can now be subjected to protein evolution campaigns to improve its activity.
A fully functional protocol has been developed and optimized for the creation of Polyethylene nanoparticles that are capable of stable suspension in aqueous solution. When encapsulated in microfluidic picolitre droplets, the concentration of these nanoparticles can be directly measured using a light scattering microfluidic rig. This assay can be implemented to directly measure the PE degrading activity of enzymes in a ultra-high throughput manner.
Nine bacterial strains capable of growth on Polyethylene have been isolated from environmental sources, and potential PE degrading enzymes have been identified on their annotated genomes. In addition, at least one PE degrading enzyme has been identified by screening genomic libraries for the capacity to confer growth on PE as a sole carbon source in alternative host strains.
A transcription factor evolution platform has been developed to allow for fast, efficient and accurate evolution of transcription factors to recognize alternative small molecule activators.
These results are in preparation for publication in two upcoming scientific papers.
The capacity to screen protein libraries directly for their degradative capacity on PE is invaluable for future directed evolution campaigns on PE. Circumventing the necessity of an analogous substrate that has a fluorescent or colorimetric output will allow for direct detection of PE degradation. The field of PE biodegradation is still very much in its infancy, and the identification of novel strains capable of growth on PE as a sole carbon source, as well as their relevant proteins, improves the scientific community’s knowledge in this area allowing the identification of future directions. A transcription factor evolution platform is a helpful tool for academic and industrial endeavours, creating the possibility for rapidly developing the investigation and utilization of a vast arrange of small molecules, and for any reaction that creates such a molecule.
Overview of biological degradation of PE