Project Description
Recent research has uncovered mutations that increase the activity of PETase, an enzyme from Ideonella sakaiensis that degrades polyethylene terephthalate (PET) [1]. However, PETase activity is restricted to ambient temperatures, which may prohibit its adoption by industrial plastic degradation programs. In 2014, the iGEM Heidelberg team demonstrated that enzyme circularization via N- and C-termini joining enhances enzymatic thermostability. Combining newly discovered catalytic mutants that enhance PETase plastic-degrading activity with protein circularization thus presents a novel approach for transferring this enzyme to industry. Expression of the circularized PETase in Escherichia coli with tags for extracellular secretion via the Sec pathway further circumvents the use of poorly-characterized I. sakaiensis as a microbial chassis. Furthermore, concomitant expression of MHETase in E. coli (the second enzyme in the plastic degradation pathway of I. sakaiensis) allows for the breakdown of mono-2-hydroxyethyl terephthalate (MHET) monomers produced during PET hydrolysis into environmentally-harmless terephthalic acid (TPA) and ethylene glycol (EG) outside the cell. Ultimately, this approach offers the possibility of closed-loop recycling, as TPA and EG are valuable feedstocks for other industrial processes, including plastic production. These can be harvested from bioreactors containing E. coli secreting circularized PETase.
We are attempting to computationally-optimize PETase’s thermostability and catalytic activity through targeted mutagenesis. The mutations are chosen via computational methods such as machine learning and rational algorithm design. The thermostability mutations, in particular, are chosen based on their maximization of hydrophobicity with the constraint of active site functionality. In order to optimize catalytic activity, we will utilize transfer learning to represent PETase’s secondary structures and then generate a unique fitness function. Finally, we plan to utilize the Metabolic Valve Enumerator (MoVE) [2] developed by the Mahadevan lab to further optimize the microbe for PET degradation.
In taking on this project, we are committed to raising public awareness of the issue of plastic waste and our synthetic biology-based solution. In order to do so, we will be speaking with a number of experts in our podcast series, hosting workshops at a summer camp, and working with other local universities to create an art gala centered around microbial plastic degradation and environmentalism. Along with the experts consulted in our podcast series, we will be working closely with experts on waste management in Toronto to receive input on the direction of our project.