Recycling to grow: cofactor conservation for sustainable phospholipid biosynthesis in synthetic cells

The research described in this thesis focuses on the construction of synthetic cells from the bottom-up, i.e., by assembling non-living components to mimic aspects of living cells and learn from them. The focus is on the metabolism of cells and on how they can produce energy and use it for important functions. We begin by making a semi-quantitative analysis of what type of minimal metabolism is needed in synthetic cells. We look at the literature and compare simple living cells (derived from parasites) with the bottom-up systems. We give an overview of all modules that will be needed at the membrane. We also make important calculations that are informative on the capacity of synthetic compartments to encapsulate the required components based on their size. Our conclusion is that synthetic cells should have a diameter of 1-2 micrometers, similar to many living microorganisms. We further focus on the growth of the cellular compartment, which is a necessary step for the cell to grow and divide. The cellular compartment can be expanded by producing new building blocks called phospholipids, a process which is energy expensive. In this thesis, we study a previously developed pathway for the production of energy and we improve it so that it can operate faster and more selectively. From our data, we are able to estimate (that is, qualitatively) how long a synthetic cell will take to grow and divide, if our pathway for energy formation is used and assuming that this is the main limiting factor. We also design, fabricate and test an experimental apparatus called continuous flow dialysis that enables us to continuously expose the synthetic cells to fresh nutrients, so that they can be always active. However, we discover that this setup does not easily allow to feed the precursors needed to make phospholipids, and for that we plan to make improvements in the future. The vesicles that produce energy are expanded to also produce a precursor of phospholipids, which is then exported and used by a second population of vesicles to produce phospholipids from the outside. In this way, we have created a communicating system, whereby one type of cells produces a signal (nutrient) and the other utilizes it for its own functions (compartment growth). This resembles what occurs in living cells that have many organelles, each one with its own specific function. Finally, we also develop pathways for the recycling of internal components that are needed for the production of phospholipids from the inside of vesicles. It is extremely important to design such recycling systems, otherwise the cell is depleted from its necessary components once they are utilized, and the activity stops. This final branch of research requires further development due to technical reasons. Finally, we summarize the next steps needed in this research field.