Cohesin-Dockerin-Based Multi-Enzyme Assembly for Efficient PET Depolymerization

Efficient depolymerization of recalcitrant polymers like cellulose and plastics requires the
synergistic performance of multiple enzymes. For the complete depolymerization of polyethylene terephthalate (PET) plastics into their constituent monomers, two enzymes- PETase and MHETase- are required. Cellulosomes, which are multi-enzyme complexes on the cell surface of many anaerobic bacteria, serve as inspiration for directed assembly of multi-enzyme clusters. Functional assembly of enzymes onto designer cellulosomes has shown improved performance over free enzyme mixtures.

To achieve complete depolymerization of PET, we aimed to assemble a multi-enzyme cluster using highly-specific cohesin-dockerin interactions observed in cellulosomes. In this work, we fused PET-degrading enzymes FAST-PETase and MHETase enzymes with dockerins from three distinct cellulolytic bacteria - Clostridium cellulolyticum, Clostridium thermocellum, and Ruminococcus flavefaciens. These fusion proteins were expressed in Escherichia coli and purified using Histag purification. Enzyme activity was subsequently tested on a soluble pNPA substrate and a solid PET film substrate, and all three enzymes were observed to be active.

These enzymes could then be successfully attached to cohesin domains on a trifunctional scaffold in displayed on the surface of a Saccharomyces cerevisiae cell. It was observed that the enzymes retained activity after binding. This work is an important step in designing multienzyme complexes for PET depolymerization.