An integrated computational framework for protein engineering and novel biocatalyst design

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Background: Plastic production has grown significantly over the last few decades and only a fifth of it is being recycled globally with less than 10% recycled in Canada. The growing demand calls for an effective and eco-friendly solution to plastic degradation. Enzyme-based biocatalysts remain an attractive, ecofriendly solution for various industrial and green chemistry applications, including the production of clean and renewable energies and bioremediation of pollutants. While beneficial, native enzymes often underperform within harsh industrial conditions which hinders the large-scale translation of biocatalysts. Therefore, the engineering of innate enzymes to optimize their physicochemical properties and catalytic potencies for suboptimal conditions is a critical avenue of research. While classic methods such as directed evolution, random mutagenesis are widely used for engineering, identifying the suitable hotspots for modification involves the sampling of vast protein sequence space and a myriad of mutations. Further, it is important to gain fundamental knowledge on the mechanisms of enzymes to develop an efficient biocatalyst. With the rapid advancements of high-performance computers and sophisticated software technologies, computational methods are currently suited to take on these challenges. Objectives: This research program aims at developing a computational framework that integrates bioinformatics, phylogenomics, advanced atomistic modelling, molecular simulation, and machine learning to understand the evolution, structure-function relationships of enzymes for guiding targeted protein engineering and biocatalyst design. My research team and I will particularly focus on engineering the polyethylene terephthalate (PET)-hydrolyzing enzymes (or P-HEs) such as PET hydrolase (PETase), monohydroxyethyl terephthalate hydrolase (MHETase), and cutinase for recycling of PET plastics. Our specific objectives are: (O1) Characterize the structures, molecular recognition mechanisms and catalyses of P-HEs (O2) Reconstruct ancestral states to understand molecular evolution and guide engineering of P-HEs and (O3) Explore substrate channeling and biochemically validate in silico-designed P-HEs. Impact: This research will provide original knowledge in basic bioscience and chemical biology by enhancing our understanding of the structure-functions of P-HEs. Our research will provide novel molecular-level insights into substrate binding, enzymatic hydrolyses of PET, hotspots for modifications, and engineered P-HEs that could be useful for developing an optimal catalyst for tackling the impending challenges from plastic waste. This program will train next-generation scientists in applying computational techniques for protein engineering. The long-term goal of this program is to develop a novel, stable and efficient biocatalyst for plastic biodegradation. Our research will also offer a versatile computational framework for designing biocatalysts.