A high-throughput expression and screening platform for applications-driven PETase engineering.

The environmental consequences of plastic waste have impacted all kingdoms of life in terrestrial and aquatic ecosystems. However, as the burden of plastic pollution has increased, microbes have evolved to utilize anthropogenic polymers as nutrient sources. Of depolymerase enzymes, the best characterized is PETase, which hydrolyzes aromatic polyesters. PETase engineering has made impressive progress in recent years; however, further optimization of engineered PETase toward industrial application has been limited by lower throughput techniques used in protein purification and activity detection. Here, we address these deficiencies through development of a higher-throughput PETase engineering platform. Secretory expression via YebF tagging eliminates lysis and purification steps, facilitating production of large mutant libraries. Fluorescent detection of degradation products permits rapid screening of depolymerase activity in microplates as opposed to serial chromatographic methods. This approach enabled development of more stable PETase, semi-rational (SR) PETase variant containing previously unpublished mutations. SR-PETase releases 1.9-fold more degradation products and has up to 7.4-fold higher activity than wild-type PETase over 10 days at 40°C. These methods can be adapted to a variety of chemical environments, enabling screening of PETase mutants in applications-relevant conditions. Overall, this work promises to facilitate advancements in PETase engineering toward industrial depolymerization of plastic waste.