Periodic Reporting for period 4 - ABCvolume (The ABC of Cell Volume Regulation) Reporting period: 2020-01-01 to 2020-06-30 Summary of the context and overall objectives of the project The bottom-up construction of synthetic cells from molecular components is arguably one of the most challenging areas of research in the life sciences. What is currently missing is a blue print for synthesizing life-like systems from molecular components. Without comprehensive instructions and design principles we rely on the construction of relatively simple reaction routes and biochemical principles to operate the essential functions of life. In our strategy the first forms of synthetic life will not make every building block for polymers de novo via complex pathways, rather they will be fed with precursors of metabolism such as amino acids, fatty acids and nucleotides. However, controlled energy supply and control over the internal pH, ionic strength, macromolecular crowding and osmotic pressure are crucial for any synthetic cell, no matter how complex. Building such systems requires control over the accumulation, formation and degradation of the interacting chemicals and homeostasis of the internal physical-chemical conditions. The provision and consumption of ATP lies at the heart of this challenge. The specific aims of the project:(i) To develop a network for cell volume regulation by synthesizing a vesicle system with sustained production of metabolic energy and capacity to build up sufficient osmotic pressure to expand, yet prevent it from lysing;(ii) To elucidate the molecular mechanism of gating and translocation of an osmoregulatory ABC transporter, a key component of the volume regulatory network, using state-of-the-art reconstitution technology and single-molecule optical microscopy. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Metabolic network for fuelIn this project, my ERC team has developed vesicle systems with sustained production of ATP from the hydrolysis of the amino acid arginine and generation of a proton motive force from the decarboxylation of malic acid, which allows metabolic energy-dependent processes such as solute transport to proceed for long-periods of time and to generate sufficient osmotic pressure to enable the vesicles to inflate and recover from imposed osmotic stress (https://youtu.be/rCCrxK8K1Ms; Figure 1). In fact, the metabolic network enables us to control the transmembrane fluxes of nutrients and to demonstrate basic physicochemical homeostasis for up to at least one day. Despite the simplicity of the network and knowledge of the individual components, emergent (futile) side reactions and complex pH behaviour were observed, which could be rationalized retrospectively. Importantly, we have demonstrated that for a well-functioning out-of-equilibrium network, the generated ATP must be used by another process, akin the situation in real living cells. The productive and futile path and the impact of varying load on the system have been modelled (with the group of Matthias Heinemann), and the mathematical analysis has guided the further designs of life-like systems. In addition, we have measured and modelled the solute permeability of the vesicles and activity of co-reconstituted transporters for different lipid compositions to meet the requirements for some of the essential functions of a cell. We find that weak acids and bases, glycerol, carbon dioxide and water permeate the membrane at sufficiently high rates without the need for active membrane transport, and a judiciously chosen combination of five lipids allows the tested transport proteins from different sources to perform at high rates.In a follow up study in collaboration with the group of Arnold Driessen we have coupled the production of ATP to the synthesis of phospholipids and cofactor regeneration; the latter is of great importance for the design of various biosynthetic reaction networks. The synthesis of lipids up to phosphatidic acid has been achieved, including the recycling of cofactors ATP, ADP, AMP, coenzyme A, glycerol-3-phosphate, pyrophosphate and inorganic phosphate. The coupling of the metabolic network for fuel to these biosynthetic process is one of the most advanced functional reconstitutions of a chemically defined network ever achieved, which allows the development of more complex life-like systems with adaptive behaviour in terms of lipid and protein synthesis, cell growth and intercellular communication.Gating of membrane transportUnlike solute permeation by passive diffusion, transport proteins allow the gated transport of molecules across the membrane, and the accumulation of nutrients and excretion of metabolic end products against their concentration gradient. A key component of our volume regulatory network is the ATP-binding cassette (ABC) transporter OpuA, which is ubiquitous in prokaryotic life and protects cells against dehydration. A high concentration of salt or sugar in the environment dehydrates microorganisms and stop them growing, a property that is used for many centuries to preserve food products. A remarkable feature of OpuA is that the transport protein is essential under some and detrimental under other environmental conditions, highlighting the necessity of a fine-tuned regulation mechanism.In natural ecosystems (micro)organisms are exposed to fluctuating conditions and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor, and is dangerous if the cell is not capable of rapidly restoring its volume. OpuA restores the cell volume by accumulating large amounts of compatible solutes such as glycine betaine. We have coupled OpuA to the metabolic network for fuel (Figure 2) and elucidated its regulation mechanism. We have found a new structural element located in the ATP-binding domain of OpuA that may act as the ionic strength sensor and allow the transporter to be gated by osmotic stress. In addition, we have found that cyclic-di-AMP can lock OpuA in a conformation that completely blocks transport, thereby serving as a safety check to prevent the unbridled uptake of compatible solutes under conditions that the cell is in danger of lysis. Strikingly, cyclic-di-AMP is a recently discovered new and crucial 2nd messenger in prokaryotes and eukaryotes, which acts as a master regulator in a variety of vital cellular processes. Our work demonstrates metabolic energy conservation and cell volume regulatory mechanisms in a cell-like system at a level of complexity minimally needed for life.Technological developmentsA number of technologies have been key for the realization of the metabolic network for fuel and the gating mechanism of OpuA, which were developed in house during the course of the project:- Fluidic device to enable a constant external environment (pH and substrate concentration, removal of end-products) (Figure 3);- Immobilization of synthetic cells for microscopy imaging;- Fluorescence-based sensors to probe physicochemical homeostasis;- Kinetic assay and mathematical model to describe solute permeation across membranes- Bioinformatic tool and protomer labelling of ABC transporters to facilitate fluorescence studies. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Progress beyond the state of the art1. Out-of-equilibrium network for fuel and physicochemical homeostasis of synthetic cells;2. Metabolic network for nucleotide-based cofactor regeneration;3. Novel gating mechanism for membrane transport to prevent the unbridled uptake of osmolytes; double brake on transport through sensing and regulation by ionic strength and nucleotide-based 2nd messenger. Figure 2 Figure 1 Figure 3
