Reconstitution of molybdenum cofactor biosynthesis in giant vesicles

Abstract

Bottom-up synthetic biology is used for the understanding of how a cell works. It is achieved through developing techniques to produce lipid-based vesicular structures as cellular mimics. The most common techniques used to produce cellular mimics or synthetic cells is through electroformation and swelling method. However, the abovementioned techniques cannot efficiently encapsulate macromolecules such as proteins, enzymes, DNA and even liposomes as synthetic organelles. This urges the need to develop new techniques that can circumvent this issue and make the artificial cell a reality where it is possible to imitate a eukaryotic cell through encapsulating macromolecules. In this thesis, the aim to construct a cell system using giant unilamellar vesicles (GUVs) to reconstitute the mitochondrial molybdenum cofactor biosynthetic pathway. This pathway is highly conserved among all life forms, and therefore is known for its biological significance in disorders induced through its malfunctioning. Furthermore, the pathway itself is a multi-step enzymatic reaction that takes place in different compartments. Initially, GTP in the mitochondrial matrix is converted to cPMP in the presence of cPMP synthase. Further, produced cPMP is transported across the membrane to the cytosol, to be converted by MPT synthase into MPT. This pathway provides a possibility to address the general challenges faced in the development of a synthetic cell, to encapsulate large biomolecules with good efficiency and greater control and to evaluate the enzymatic reactions involved in the process. For this purpose, the emulsion-based technique was developed and optimised to allow rapid production of GUVs (~18 min) with high encapsulation efficiency (80%). This was made possible by optimizing various parameters such as density, type of oil, the impact of centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume. Furthermore, the method was optimised in microtiter plates for direct experimentation and visualization after the GUV formation. Using this technique, the two steps - formation of cPMP from GTP and the formation of MPT from cPMP were encapsulated in different sets of GUVs to mimic the two compartments. Two independent fluorescence-based detection systems were established to confirm the successful encapsulation and conversion of the reactants. Alternatively, the enzymes produced using bacterial expression and measured. Following the successful encapsulation and evaluation of enzymatic reactions, cPMP transport across mitochondrial membrane has been mimicked using GUVs using a complex mitochondrial lipid composition. It was found that the cPMP interaction with the lipid bilayer results in transient pore-formation and leakage of internal contents. Overall, it can be concluded that in this thesis a novel technique has been optimised for fast production of functional synthetic cells. The individual enzymatic steps of the Moco biosynthetic pathway have successfully implemented and quantified within these cellular mimics.