Directed Evolution of Multimodal Plastic Degrading Enzymes

The inability to break down plastics is a major obstacle in the ongoing quest for sustainability. Advancements have been made in identifying enzymes that can break down certain polyesters; however, these enzymes are limited to the primary substrate and closely related plastic polymers. By increasing the catalytic promiscuity of polyester-degrading enzymes through directed evolution, a multimodal plastic degrading enzyme can be created. In order to achieve this, a polyesterase was first designed for secretion from the periplasm of E. coli. Through active site analysis, four sites were selected for amino acid randomization to create a >300,000 member mutant library. Next, a plate-based activity analysis system was developed to ensure efficient and reproducible screening of variants from the mutant library. Mutants with signs of activity on polyester- or polyurethane-containing agar were propagated and tested in liquid assays using a spectroscopic probe. A mutant was identified that retained polyesterase activity with newly developed polyurethane-degrading activity. Computational analysis indicated that these changes expanded the substrate pocket while retaining the positioning of the catalytic residues essential to the hydrolysis reaction. This provides the first demonstration of the directed evolution of a multimodal plastic degrading enzyme and serves as a model for further expanding polyesterase promiscuity to include multiple hydrolyzable plastics.