Enzyme Benchmarking with Polyethylene Furanoate Soluble Scaffolds for Directed Evolution of PEFases

Plastic waste is a major threat in our industrialized world and is driving research into bioplastics. The success of biobased polyethylene furanoate (PEF) as a viable alternative to polyethylene terephthalate (PET) of fossil origin will depend on designing effective enzymes to break it down, aiding its recycling. Here, a panel of fungal and bacterial cutinases were functionally expressed in a tandem yeast expression system based on Saccharomyces cerevisiae and Pichia pastoris. The activity of the enzyme panel was tested with soluble PEF model scaffolds, observing a correlation with the degradation of real PEF powder. A high-throughput colorimetric screening assay based on the PEF scaffold diethyl furan-2,5-dicarboxylate was developed, establishing the basis for future directed evolution campaigns of PEFases.