PETase Engineering for Enhanced Degradation of Microplastic Fibers in Simulated Wastewater Sludge Processing Conditions

Microplastic accumulation is an issue of growing environmental importance for which enzymatic biodegradation has been proposed as a remediation strategy. Because treated wastewater products are one of the major conduits by which microplastics enter ecosystems, the wastewater treatment system is a potentially impactful intervention point for microplastic biodegradation. PETase is a polyester-degrading enzyme with great promise for wastewater treatment applications. While PETase has been engineered for enhanced thermostability in defined, buffered conditions, previous research has not explored improving its chemostability, an important factor to consider in targeted PETase applications due to the tendency of microplastics to accumulate in complex and variable environments such as sewage sludge. We show that tailoring screening conditions to chemically simulate sludge processing enables high-throughput measurement of a semi-rational PETase mutant library. Combinatorial mutagenesis yields a triple mutant, "Sludge-PETase", with up to 17.4-fold enhanced activity in simulated sludge conditions. To our knowledge, this study is the first to demonstrate that targeted mutagenesis can increase PETase chemostability. The work presented makes substantial progress toward PETase intervention in wastewater treatment facilities. Further, the application-driven approach taken in this study to engineering PETase represents an important strategy for engineering proteins to perform in diverse, complex systems.