Engineering Proteins for Medical and Industrial Applications: From Neuroscience to Environmental Remediation
Creators: name: Vongsouthi, Vanessa
Publisher: name: The Australian National University
Year: 2024

description: Protein engineering is a discipline dedicated to manipulating the intricate relationship between protein sequence, structure, dynamics, and function, to yield biomolecules with desired properties for applications across a diverse range of industries, including pharmaceuticals, synthetic biology, and biomanufacturing. The endeavour of protein engineering involves navigating the expanse of protein sequence space, where even a modest protein of 100 amino acids has 20^100 possible sequence combinations. Given that the enormity of protein sequence space far exceeds the practical limitations of protein characterisation, a fundamental challenge of protein engineering is optimising methods of sequence exploration to efficiently discover proteins with novel or enhanced functionality. In the works presented in this thesis, the applied protein engineering methods span across rational design, sequence-guided structure-based design, ancestral sequence reconstruction, and consensus design. In particular, these methods are applied to two main areas of research: the development of fluorescence-based optical biosensors for neurotransmitters, and the enhancement of plastic-degrading enzymes for industrial applications. By applying these techniques, we successfully engineer fluorescence-based optical biosensors for the detection of the transmitter molecules, D-serine and glycine, providing tools to study the mechanisms of learning and memory in-vitro and in-vivo. Simultaneously, we apply protein engineering tools to the evolutionary exploration and enhancement of polyethylene terephthalate (PET)-degrading enzymes from Ideonella sakaiensis and the cutinase family, identifying enzyme variants with increased stability and activity. In Chapter 1, the protein engineering methods and protein systems of particular interest to this thesis, i) fluorescence-based optical biosensors and ii) plastic-degrading enzymes, are introduced. In Chapter 2, we review the structural and evolutionary-based approaches towards engineering genetically encodable small-molecule biosensors. This review provides an overview of the fundamental concepts relevant to Chapter 3, where we present the engineering of genetically encoded biosensors for both D-serine and glycine, demonstrating the construction of novel biosensors and their subsequent in-vitro and in-vivo application to study transmitter dynamics. Chapter 4 shifts to the engineering of plastic-degrading enzymes, providing a review on the application of directed evolution to enhance such enzymes for industrial use. In Chapter 5, we present the application of multiplexed ancestral sequence reconstruction to the exploration of the sequence-fitness landscape of cutinases with PETase activity, revealing a rugged landscape and uncovering ancestral cutinases with improved PETase activity relative to extant homologues. Similarly, Chapter 6 applies ancestral sequence reconstruction to the study of PETase from Ideonella sakaiensis, providing insights into the evolution of PETase activity and characterising ancestral PETases with improved stability and activity. Building on this, Chapter 7 applies a novel activity assay and consensus-based design to the engineering of mono(2-hydroxyethyl) terephthalate (MHET) hydrolase (MHETase), the second enzyme involved in the PET degradation pathway of I. sakaiensis, identifying variants with improved soluble expression and whole-cell activity compared to the wild-type enzyme. Lastly, the thesis is concluded by a brief discussion on the future research directions of engineering genetically encoded biosensors and plastic-degrading enzymes.