Applications of Systems-Driven Protein Engineering Toward In Situ Water Safety Technologies

Abstract

Engineered proteins (defined here as proteins with a non-native amino acidsequence and/or expression system) are a remarkably versatile tool whose inherent plasticity enables targeting toward diverse applications. While numerous sectors have adopted engineered proteins for use as biocatalysts, therapeutic agents, and advanced materials, water distribution, purification, and treatment systems have been more reticent, likely due to the costs and regulations associated with engineered proteins. Nonetheless, engineered proteins have shown great promise for water safety applications. This dissertation presents a systems-driven approach to protein engineering that aims to overcome barriers to adoption in water systems. Chapter 1 provides an overview of the importance of water safety, engineered proteins, challenges associated with application of engineered proteins in water systems, and opportunities for a systems-driven approach to overcome said challenges. Chapter 2 presents a bacteriophage-based assay for detection of a <10 cfu E. coli in 100 ml drinking water. Click conjugation of bacteriophage T4 to magnetic nanoparticles is mediated by site- specific incorporation of alkyne groups into a T4 capsid protein using artificial amino acid mutagenesis. Magnetic separation of the target bacteria is mediated by the immobilized phages, increasing the efficiency of the assay. Chapter 3 reviews the literature on microplastics in food and agriculture and suggests in situ biodegradation in wastewater treatment plants as a potential solution to the microplastic problem. Chapter 4 focuses on the development and validation of a high-throughput engineering platform for PETase, a plastic-degrading enzyme. A semi-rational library of mutants is constructed and screened iteratively to generate SR-PETase, which has up to 7.4-fold higher activity than its wildtype counterpart. Chapter 5 applies this platform to engineering PETase for optimal activity in sewage sludge conditions. The same mutant library is screened and Sludge-PETase, with up to 17.4-fold higher activity than wildtype in sludge conditions is presented. Chapter 6 focuses on immobilization of PETase to mesoporous silica nanoparticles, which is found to increase activity 6.2-fold in synthetic effluent conditions and 2-fold in synthetic influent conditions. To conclude, Chapter 7 highlights important points made by chapters 2, 4, 5, and 6 and suggests future work for each.