BIODEGRADE focused on building a modified microbial platform for PET breakdown and upcycling, with a strong emphasis on protein engineering. Our interdisciplinary approach combined computational design with laboratory synthetic biology experiments.
1. Computational protein engineering of PET-degrading enzymes: We successfully engineered novel catalytic sites within native E.coli proteins, enabling them to degrade PET. We called these designed proteins plurizymes, proteins that gained new PET-degrading activity through the insertion of a catalytic site, while retaining their original functions. Our approach used a multi-stage computational pipeline. First, we systematically scanned the entire E. coli genome to select suitable protein candidates, using AI tools to find the localization of the protein and if they are inserted on the membrane, prioritizing soluble and extracellular proteins. We did this because these proteins will have a higher probability to came in contact with PET particles or molecules. Next, we used AlphaFold2 to predict and generate the high quality 3D structures of these proteins. We then applied the molecular modeling software PELE (Protein Energy Landscape Exploration) to scan their surfaces for potential PET binding sites, using the PELE Global Exploration protocol. We used 3 different substrates at these stage, using PET substrates molecules of different lenght (different number of polymers in the chain). We selected the proteins that bind at least two of the substrates in the same binfing site with high affinity. In the fourth stage, we designed catalytic triads, based on wild-type PETase active sites (serine, histidine, and aspartic acid), near these binding pockets. We computationally evaluated and ranked mutant variants using PELE-induced fit simulations, focusing on interaction energy and spatial arrangement of triad residues, that should fulfill a series of distances to be considered in a catalytic position. Finally, molecular dynamics simulations were used to refine and identify the most promising plurizyme candidates, based on metrics that describe the reaction of the enzyme.
2. Experimental target validation in vitro: we produced and tested the selected enzymes in the laboratory using PET building blocks monomers. Three designed proteins showed PET-degrading activity, with two being particularly effective. These were further tested on PET powder and PET nanoparticles and two display high activity with both substrates.
3. Engineering E.coli for efficient degradation of nanosized PET and utilization as carbon source: Using CRISPR-Cas9 gene editing technology, we reprogramed the E.coli genome and exchange the native gene with our most promising plurizyme, with remarkably only 3 point mutations compared with the native. This created a new E.coli strain able to break down PET nanoparticles (smaller than 5 nm) into its constituent monomers ethylene glycol (EG) and terephthalic acid (TPA), a new function that did not harm the bacteria's growth. We then showed that, by introducing genes for EG metabolism, this strain could use one of the PET degradation products, EG, as a nutrient source for growth. Indeed, the engineered bacteria could not only degrade PET nanoparticles, but also grow using the resulting EG as carbon source.
4. Development of E.coli for the bioconversion of nPET waste into valuable compounds: We further engineered our plastic-degrading E.coli to convert the other PET breakdown product, TPA, into a valuable substance, protocatechuic acid (PCA). We introduced additional genes enabling this conversion. When this engineered strain was given only PET nanoparticles, it effectively broke them down, used the released EG for growth, and produced PCA from the TPA. This demonstrated the strain's ability to both degrade PET nanoparticles and convert its breakdown products into a nutrient and a valuable biochemical.
By the project's end, BIODEGRADE successfully created a proof-of-concept microbial system for enzymatic PET degradation using their own native proteins, and the upcycling of its breakdown products. These results provide a strong foundation for developing new and eco-friendly biotechnological solutions for plastic waste treatment.
