ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis

The bottom-up construction of an autonomously growing, self-reproducing cell represents a great challenge for synthetic biology. Synthetic cellular systems are envisioned as out-of-equilibrium enzymatic networks encompassed by a selectively open phospholipid bilayer allowing for protein-mediated communication; internal metabolite recycling is another key aspect of a sustainable metabolism. Importantly, gaining tight control over the external medium is essential to avoid thermodynamic equilibrium due to nutrient depletion or waste buildup in a closed compartment (e.g., a test tube). Implementing a sustainable strategy for phospholipid biosynthesis is key to expanding the cellular boundaries. However, phospholipid biosynthesis is currently limited by substrate availability, e.g., of glycerol 3-phosphate, the essential core of phospholipid headgroups. Here, we reconstitute an enzymatic network for sustainable glycerol 3-phosphate synthesis inside large unilamellar vesicles. We exploit the Escherichia coli glycerol kinase GlpK to synthesize glycerol 3-phosphate from externally supplied glycerol. We fuel phospholipid headgroup formation by sustainable l-arginine breakdown. In addition, we design and characterize a dynamic dialysis setup optimized for synthetic cells, which is used to control the external medium composition and to achieve sustainable glycerol 3-phosphate synthesis

Building a community to engineer synthetic cells and organelles from the bottom-up

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives

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

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells:A Membrane Perspective

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.</p

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.</p

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell

Building a community to engineer synthetic cells and organelles from the bottom-up

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.BN/Marileen Dogterom La