21ENGBIO: Engineering novel amyloid biofilm based material for capture and degradation of micro-plastics

Plastic pollution, whereby plastic objects and particles are accumulated is a current, pressing, and costly environmental problem both in the UK and worldwide. Polyethylene terephthalate (PET) is the chemical name for polyester, a common type of plastic which would take up to 800 years to degrade on its own. PET waste also tends to be converted by abrasion into microplastics, which are known to have adverse effects on animals and plants. Recently, researchers have discovered an enzyme called PETase which can actively degrade PET into non-harmful substances. Currently, the application of PETases to plastic degradation is limited by the requirement to deploy PETases in solution. We propose combining PETases with a meshed biomaterial formed from amyloid fibrils to form a novel material. This material can be exported via bioengineered cells to generate a non-toxic mesh with malleable properties for potential use in large scale designs to remove and to degrade plastic waste.

Amyloid fibrils are a type of protein structures with a characteristic shape. Some amyloid fibrils are known to be associated with diseases such as Alzheimer's disease, Parkinson's disease, and systemic amyloidosis. However, many amyloid forming are beneficial, or "functional" in that they fill essential biological roles. Bacteria such as E. coli have been found to utilise amyloid fibrils made from the curli protein to form a protective biofilm as the amyloid fibrils form a hard to pass through mesh which protects the E. coli against viral infection. To do this, the E. coli cells naturally contain a system which forces the amyloid fibrils to form outside the cells in a controlled manner by sticking individual curli proteins together as they are pushed out of the cell. It is already possible to hijack this system to export amyloid fibrils made from different amyloidogenic proteins, for example another functional amyloid protein from yeast called Sup35.

Sup35 forms amyloid fibrils in yeast cells and normally is then used as a messenger to other yeast cells. Importantly, Sup35 has three parts and only two of these are required to make the amyloid fibrils, these two parts are known as Sup35NM. We intend to replace the 3rd part with enzymes such as PETase which could functionalise the Sup35NM amyloid fibrils and imbue them with PET degrading functionality.

In this project, we intend to bioengineer E. coli cells to generate and export a biofilm containing the novel material of Sup35NM amyloid fibrils decorated with PETase and other plastics degrading enzymes. This will generate a robust yet malleable, non-toxic substance which could be applied to devices such as filters or even 3D printed onto complex designs with the ability to degrade plastics. Since neither plastics nor amyloid interact well with water, the amyloid mesh will likely be able to capture and to improve the contact between the plastic degrading enzymes and the plastic particles. Furthermore, the very high density of enzymes that can be achieved in an amyloid mesh will further improve plastic degradation, and the ability to combine different types of enzymes may improve the total plastic degradation activity. The proposed amyloid mesh, therefore, could offer an environmental solution to the problem of plastic pollution.

To produce a novel amyloid mash material decorated with PET degrading enzymes, we will first produce chimera protein monomers using the pET/BL21 protein expression system. The chimera proteins will consist of the amyloid forming part of the yeast protein Sup35 (called Sup35NM) linked to PETase, MHETase or BHETase. These three enzymes will provide Sup35NM amyloid fibrils with enzymatic activities that catalyse a series of reactions that breaks down PET into ethylene glycol and terephthalic acid. Secondly, we will make constructs containing the three catalytic chimera proteins for expression and extracellular export into the media as monomers, where they can assemble in situ into large fibril networks, using the Curli-dependant amyloid generator (C-DAG) system. We will also assemble the chimera proteins into amyloid fibril networks decorated with PET degrading enzymes in vitro, which we will confirm by kinetics and AFM imaging experiments. Finally, we will assess the structure and the PET degrading activity of the fibril mesh material formed in vitro and in situ. Thus, this projeect will produce a novel biomaterial which is a robust yet malleable, non-toxic substance that could act as microplastics capturing and degrading filters.