Creating and functionalising a synthetic compartment in plants

Compartmentalisation is a universal feature of life, allowing organisms to separate their functions from each other and the environment. It enables greater control and complexity for cellular processes. Taking a cue from nature, engineered compartmentalisation has become one of the top priorities of synthetic biology. While many alternative strategies have emerged, building a synthetic membranous organelle de novo remains prohibitively difficult, despite its potential as a tool for metabolic engineering. This lack of an orthogonal synthetic organelle especially hampers the development of the nascent plant molecular farming industry. In this thesis, I describe the design and characterisation of a novel synthetic membranous compartment derived from the plant endoplasmic reticulum (ER), and proof-of-principle experiments to showcase its potential as a bioproduction platform in plants. The formation of the compartment is triggered by overexpression of a single recombinant scaffold protein, and I investigated the relationship between the structure of the scaffold protein and the morphology of the resultant compartment. I also compared the compartment to a conceptually similar ER rearrangement called organised smooth endoplasmic reticulum to determine their relation. Notably, the formation of the compartment had very little impact on the overall health of the host plant, and successful pilot experiments for the purification of the scaffold and other proteins anchored to it, as well as running enzymatic reactions on the surface of the compartment demonstrate the great potential of this system to serve as a plant synthetic biology tool. With further development, this platform could be a valuable asset to the plant biosynthesis and recombinant protein production industry.