Since its creation, plastic has grown in its world-wide use. Its versatility makes it a very commonly used material, particularly in the case of polyethylene terephthalate (PET) plastics, used to make bottles, clothing materials and food containers. But this same versatility and resistance to erosion also make it all the harder to recycle. Only 9% of all plastic ever made has been recycled, due to a lack of technologies and unfavorable economics, 12% is incinerated and 79%, often scattering into the natural environment or into the sea. While recycling these plastics is crucial, often the resulting product has a much lower value than the original plastic undermining any economic viability in this goal. Upcycling is the process of transforming a product, usually waste or simply unwanted, into something of higher value, and it is crucial to assure economic interest in plastic degradation, as ensuring that the degradation of these pollutants can also increase their value can be more appealing. Recent developments in the field of enzymatic plastic-degradation has created interest into these as a way of reducing the effects of plastic pollution. We decided to focus on PET plastic degradation, due to its abundance and as it has the largest number of characterized enzymes capable of acting upon it. The goal of this project was, then, to develop a single bacterium model of E. coli with a fully integrated metabolic pathway, including PETase, MHETase, LCC, the TPADO enzymatic complex and a TPA transporter. This would allow for PET degradation into protocatechuic acid (PCA), a valued pharmaceutical metabolite. This metabolic pathway can be illustrated as below.

To create a fully integrated pathway in E. coli, we first needed to express each enzymes in E. coli. The general workflow we aimed for was the following, and was attemped for all enzymes/proteins:

1. Design construct - Include CDS for the enzyme/protein and combine it with an RBS, T7 promoter and terminator, lac operator, a C-terminal His-tag and BioBrick prefix and suffix.
2. Clone construct into E. coli - Into DH5α cells for amplification and later on BL21 cells for protein (over)expression.
3. Protein overexpression - Under the lac operator with T7 RNA polymerase.
4. Purification of protein - With e.g. IMAC and gravitraps.
5. Possibly uplscale production in bioreactors - At Testa Center's facilities

The experiments used for the different general parts of the workflow are descirbed in General protocols. In addition to the enzymes and proteins mentioned earlier, leaf-branch compost cutinase (LCC) was also attempted to be integrated into the E. coli expression system. LCC could be used as an alternative to PET degradation by PETase, but also in a high temperature pretreatment of PET. When it comes to the work with the TPA transporter, it was based on the same workflow but based on different promoter system and without BioBricks.

The success of the different proteins varied, but the work with each were nonetheless important and educational. To find out more about what work was done with each enzyme and protein, see the repective page for each enzyme (in the dropdown menu).

In addition to our wet lab work, we also decided to investigate more about the last enzyme in the pathway, DCDDH, since it was not as well known as the others. Therefore, our modeling group worked with getting some more insights over DCDDH and look into the possibility of producing new variants of it to improve its catalytical activity. In our modeling, programs such as CavityPlus, DockThor, MODELLER, GROMACS and Alphafold 2.0 were used, together with an AI approach with Generative Adversarial Neural Network (GANN) called ProteinGAN. More details of our modeling work can be found under in Modeling.