Bott, Hannah2025-08-122025-08-12https://hdl.handle.net/1885/733767313Proteins can be powerful tools for a myriad of applications; however, their uses are limited unless they can be engineered to purpose. Evolutionary methods of engineering proteins include ancestral sequence reconstruction (ASR) and directed evolution. ASR can be an effective method to study the sequence-function relationship over time. Additionally, ASR can predict thermostable ancestors that may have improved expression as well as improved temperature and solvent stability for future applications. Directed evolution can be useful for engineering a protein with a desired property as it samples a large sequence-function space. Whilst both ASR and directed evolution have been commonly used to engineer proteins for applications in the pharmaceutical, bioremediation and green chemistry fields, amongst others, their potential in understanding transcription factors and for enzyme-mediated radical polymerisation (EMRP) has been relatively underexplored. In this thesis, the utility of evolutionary methods in these fields was investigated using various biochemical techniques and material characterisation methods. In Chapter 2, ligand binding of ancestrally reconstructed proteins of the lac repressor (LacI) family was characterised in detail to reveal an enthalpic-entropic trade-off in ligand binding along the LacI trajectory and increasing specialisation towards B-substituted D-galactosides as ligands. It was found that functional changes in ligand binding between two ancestors result from complex and likely epistatic interactions between residues. Hence, this research could inform future engineering efforts of LacI family transcription factors. Also, as the genotype-phenotype landscape of the LacI family of transcription factors has proved difficult to characterise in the past, this study of the LacI family highlights the ability of ASR to characterise the sequence-function landscape in a 'smart' way, thereby enabling deeper insights for future protein design. ASR was further utilised in Chapter 3 to identify an ancestral horseradish peroxidase (HRP) with improved recombinant expression. HRP is a commonly used enzyme, however, its use is restricted because it is difficult to express recombinantly and plant-derived HRP has batch-to-batch variation. Due to these limitations, ASR was utilised to engineer HRP for improved recombinant expression. It was found that ASR successfully identified ancestral peroxidases that could be expressed recombinantly in E. coli in a soluble, active form. Selected ancestral peroxidases were demonstrated to have relatively high thermostability whilst still possessing the desired peroxidase activity. It was also shown that one of the ancestral peroxidases could replace HRP in the EMRP of poly(N-isopropylacrylamide) (PNIPAm). Chapter 4 further explores the use of evolutionary engineering methods in EMRP. Previously, HRP has commonly been used in EMRP of materials like alginate/PNIPAm. However, due to the limitations of HRP, it was investigated whether a peroxygenase engineered for high recombinant yields using directed evolution could be utilised instead. It was found that this peroxygenase, unspecific peroxygenase PaDa-I (UPO), could replace HRP in the formation of alg/PNIPAm to form a hydrogel with similar printability, thermosensitive properties and relatively similar mechanical properties to the HRP-formed alg/PNIPAm. Thus, the potential of UPO for use in EMRP reactions was established for the first time. It was then demonstrated that UPO-formed alg/PNIPAm containing K. phaffii or S. cerevisiae could act as a producer of eukaryotic proteins, thereby forming a 3D-printable, thermosensitive, engineered living material bioreactor. Overall, this thesis highlights the utility of evolutionary methods in gaining further insights into protein genotype-phenotype landscapes and in engineering proteins for enzyme-mediated polymerisation applications.en-AU202510.25911/2B4Q-C003