As contribution for future iGEM Teams which want to tackle the problem of plastic pollution, we added new litterature information to the PETase part BBa_K2010999. It describes the mutations that lead to the fastest and most stable PETase mutant engineered so far.

Poly(ethylene terephthalate) hydrolysing enzymes like PETase with the ability to depolymerise this highly abundant plastic type gained major interest after their discovery as they posess great potential of green and scalable way of recycling even in industrial scale. Major drawbacks so far have been their low activity, instability to pH and temperature ranges and inability to use untreated PET(Taniguchi, I. et al. 2019). To overcome those problems engineering work on the enzyme PETase originally found in ideonella sakaiensis (Yoshida, S. et al. 2016) was documented within the iGEM competition as well as in scientific journals.

One of the latest Nature publications working with PETase used a machine learning-aided engineering approach with the goal to improve the overall enzyme stability (Lu, H., Diaz, D.J., Czarnecki, N.J. et al. 2022). In total they found 4 mutations S121E, T140D, R224Q and N233K which highly increased the stability both individually and in combination. They further used these findings to evaluate the hydrolytic activity of their newly generated stable mutants.

In all their generated mutants they identified one mutant called FAST-PETase (functional, active, stable and tolerant PETase) which had the highest overall activity at 50°C compared to all other known PET hydrolysing enzymes. FAST-PETase which contained the mutations S121E, D186H, R224Q, N233K and R280A performed well compared to other known enzymes in temperatures between 30, 40 °C and a pH range of 6.5-8 which represent moderate conditions which should be relatively easy to sustain on larger scale. Structural analysis revealed that the increased stability of this mutant as being due to the N233K mutation which places a positively charged lysine residue next to a negatively charged glutamic acid which results in a salt bridge. The R224Q mutation allows the glutamine to form a hydrogen bond to the carbonyl group of a neighboring serine. Finally the S121E mutation allows a new established water-mediated hydrogen-bonding network with a histidine and asparagine.

Throughout our work this summer, we also designed and synthesized a plethora of new basic parts and composite parts. Many of these parts have been adapted from up to date publications regarding PET plastic degradation, designed to fit the BioBrick assembly standard and further optimized for use in E. coli expression systems, you can find an overview of our parts here.

Lu, H., Diaz, D.J., Czarnecki, N.J. et al. Machine learning-aided engineering of hydrolases for PET depolymerization. Nature 604, 662–667 (2022). https://doi.org/10.1038/s41586-022-04599-z

Taniguchi, I. et al. Biodegradation of PET: current status and application aspects. ACS Catal. https://doi.org/10.1021/acscatal.8b05171 (2019).

Yoshida, S. et al. A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351, 1196–1199 (2016).