Reprogramming bacterial nanocompartments into light-triggered nanoreactors

Encapsulins are protein-based nanocompartments found in 1-4% of known prokaryotes, which have a set of distinct physical and functional features that make them attractive as unique reaction chambers. They self-assemble from identical protein subunits into hollow spherical structures that are 18-44 nm in diameter and exhibit good colloidal properties and robust stability. During self-assemble, encapsulins selectively package and protect cargo proteins (native or foreign) tagged with a unique encapsulation signal peptide (ESig), offering an interchangeable system for the programmed encapsulation of ESig-tagged proteins. In addition, surface pore openings allow small molecules, like cellular oxygen, to enter the internal cavities of encapsulins, facilitating their interaction with the protein cargo. The outer surfaces of encapsulins are also highly adaptable and can be genetically and/or chemically modified to further enhance their functionalities. In this thesis, I aimed to reprogram encapsulin nanocompartments to have light-activatable properties and functionalities that lend themselves to practical applications in biotechnology and biomedicine. The Fluorescent proteins KillerRed (KR) and mini-Singlet Oxygen Generator (mSOG) are unique biological photosensitizers that produce reactive oxygen specials (ROS) when irradiated with light at specific wavelengths. I reprogrammed the native function of encapsulin (Enc) from the bacterium Thermotoga maritima by loading it with ESig-tagged KR or mSOG variants. All photosensitizer-loaded Encs were recombinantly produced in Escherichia coli, purified by chromatographic methods and were found to be ~25-30 nm in size, monodisperse and fluorescent. The red fluorescent protein KR is a Type I photosensitizer that generates mainly superoxide ion (O2 Encapsulins are protein-based nanocompartments found in 1-4% of known prokaryotes, which have a set of distinct physical and functional features that make them attractive as unique reaction chambers. They self-assemble from identical protein subunits into hollow spherical structures that are 18-44 nm in diameter and exhibit good colloidal properties and robust stability. During self-assemble, encapsulins selectively package and protect cargo proteins (native or foreign) tagged with a unique encapsulation signal peptide (ESig), offering an interchangeable system for the programmed encapsulation of ESig-tagged proteins. In addition, surface pore openings allow small molecules, like cellular oxygen, to enter the internal cavities of encapsulins, facilitating their interaction with the protein cargo. The outer surfaces of encapsulins are also highly adaptable and can be genetically and/or chemically modified to further enhance their functionalities. In this thesis, I aimed to reprogram encapsulin nanocompartments to have light-activatable properties and functionalities that lend themselves to practical applications in biotechnology and biomedicine. The Fluorescent proteins KillerRed (KR) and mini-Singlet Oxygen Generator (mSOG) are unique biological photosensitizers that produce reactive oxygen specials (ROS) when irradiated with light at specific wavelengths. I reprogrammed the native function of encapsulin (Enc) from the bacterium Thermotoga maritima by loading it with ESig-tagged KR or mSOG variants. All photosensitizer-loaded Encs were recombinantly produced in Escherichia coli, purified by chromatographic methods and were found to be ~25-30 nm in size, monodisperse and fluorescent. The red fluorescent protein KR is a Type I photosensitizer that generates mainly superoxide ion under green/yellow light irradiation. Upon activation with green light, KR-loaded Enc produced similar amounts of ROS as free KR, while unloaded Enc produced no ROS. These results show that KR can be packaged inside Enc without affecting its photosensitizing functions. Alternatively, the green fluorescent flavoprotein mSOG is a Type II photosensitizer that produces singlet oxygen upon blue light irradiation. mSOG variants, mSOG1 and mSOG2, previously engineered for enhanced 1O2 generation were loaded into Enc. All mSOG-loaded Encs produced measurable quantities of 1O2 under blue light activation, with a mSOG1-loaded Enc variant (Enc-mSOG1-EsigT) shown to be the most effective. Based on these findings, the capacity for Enc-mSOG1-ESigT to trigger photosensitized reactions was evaluated in a cellular model of lung cancer. Enc-mSOG-ESigT displayed no cytotoxicity in the dark, however, when activated with blue light, it caused a ~34% reduction in cancel cell viability. Thus, this work represents the first-time protein-based nanocompartments have been loaded with functional biological photosensitizers and shown the ability to act as light-triggerable 'nanoreactors'. Furthermore, their capacity to induce phototoxicity against cancer cells highlights their potential as an exciting new nanoplatform for the photodynamic therapy of cancer. I also present preliminary work aimed at incorporating a light-triggered disassembly/reassembly mechanism into encapsulin for the loading and/or releasing its cargo. To achieve this function, I implemented a pH disassembly/reassembly approach to test the encapsulation of small-molecule drugs into T. maritima encapsulin. Additionally, protein modelling was used to identify three residues located at the interfaces of encapsulin subunits, which could be substituted with photo-responsive unnatural amino acids (UAA) (e.g. azobenzene) to potentially mediate light-triggered diassembly/reassembly of the nanocompartment -- abstract.