title: Enzyme-mediated radical polymerization for intracellular and solution reactions
Creators: name: Ornati, Eleonora
Publisher: name: UNSPECIFIED
Year: 2025

description: Protein-mediated polymerization represents a rapidly expanding field at the interface between biotechnology and materials science, combining the catalytic function of proteins to the formation of polymeric structures under mild, biological conditions. More recently, intracellular polymerization has raised great interest because of its underlying potential to modify and functionalise living organisms. Through intracellular polymerisation, living cells' behaviour can be modulated to achieve cryostasis, tumour-specific targeting, or vaccine development. Furthermore, cells can be fixed to study or modify the bio-membranes, expose pathogenic peptides for T cell expression or constitute the base for regenerative medicine. Initial efforts to achieve intracellular polymerization focused on biorthogonal chemical reactions to avoid cell death or destruction, maintaining cell structures and functions unaltered. 
In this thesis, a further investigation of protein-mediated radical polymerization and its implementation into intracellular polymerization strategies in prokaryotic cells was conducted. Starting from previous knowledge about protein-mediated ATRP-like reactions in aqueous solutions, a novel application in intracellular polymerisation has been investigated. The well-known bacteria Escherichia coli BL21 has been chosen as a model organism for this purpose. Surprisingly, E. coli has demonstrated intrinsically pro-polymerisation properties, being able to autonomously polymerise a variety of monomers under anaerobic conditions and the presence of a classical water-soluble ATRP initiator. This new ability has been used to achieve a fast and effective cytoplasmatic polymerisation in living bacteria cells that show high biocompatibility and led to an unchanged proliferation capability of polymerised E. coli cells. 
The intrinsic ability of E. coli to autonomously trigger polymerisation reactions through ATRP initiators raised the question about the biocatalyst responsible for the activity. Through a series of molecular biology and proteomics techniques, the responsible catalyst has been revealed to be the enzyme succinyl ornithine transaminase. This discovery is the first example of a non-metal-based protein catalyst able to trigger an ATRP-like reaction in aqueous and complex media, showing a direct catalytic dehalogenation activity on alkyl halide initiators. 
On the topic of non-metal-based catalysts for radical polymerisation, another enzyme was found to have an unexpected catalytic activity. Glucose oxidase can indeed start a polymerisation reaction with a direct activity on certain monomer structures, producing free radicals that can start the polymerisation, without the need for initiators or metals. This finding enriches the knowledge of this versatile flavoprotein already used as a mediator for polymerisation reactions under mild and open-to-air conditions. 
Finally, some other open projects are briefly discussed, which can be linked to the main topics addressed in this thesis. Specifically, protein-mediate RAFT polymerisation in complex solutions derived from cellular lysis and the polymerization ability of yeast cells remains an open topic that requires further investigation. 
Together, these results further enriched the knowledge of protein-initiated polymerisation and intracellular polymerization. This work not only characterizes the catalytic machinery responsible for polymer formation in E. coli but also proposes new catalytic activity to well-known enzymes. Ultimately, this work deepens our understanding of enzyme-mediated polymerization and opens new avenues for the implementation of living cells as bio factories for next-generation materials.