For energy supply to biomimetic constructs, a complex chemical energy-driven ATP-generating artificial system was built. The system was assembled with bottom-up detergent-mediated reconstitution of an ATP synthase and a terminal oxidase into two types of novel nanocontainers, built from either graft copolymer membranes or from hybrid graft copolymer/lipid membranes. The versatility and biocompatibility of the proposed nanocontainers was demonstrated through convenient system assembly and through high retained activity of both membrane-embedded enzymes. In the future, the nanocontainers might be used as a platform for the functional reconstitution of other complex membrane proteins and could considerably expedite the design of nanoreactors, biosensors, and artificial organelles.

Cytochromebo3ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitutebo3oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min−1. To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

cell's total energy budget. [2] The universal energy currency that is used for these purposes and can be found in all forms of life is adenosine triphosphate (ATP). A vast majority of the cellular energy demand is covered by either converting a large variety of energy-rich substrates into ATP in a process called oxidative phosphorylation, or by converting light (electromagnetic) energy into ATP in a process called photophosphorylation (or photosynthesis).These naturally existing energy conversions are valid in in the context of bottomup synthetic biology as well. In the latter, an artificial cell is envisioned as a compendium of functional modules, each hand-tailored to partially or entirely mimic one of the essential life processes, such as reproduction, growth, motility, etc. [3] Like their natural counterparts, all these reenvisioned synthetic processes are energy demanding, therefore, the deliberate design of synthetic cells should involve suitable energy management strategy, by, for example, continuous regeneration of ATP. Apart from the importance in the context of artificial cells, new energy conversion strategies can be considered as a standalone feature for enzymatic and cell-free biotechnology, wherein bottom-up synthetic biology might also deliver new and sustainable solutions.The notion of synthetic in terms of engineered and/or non-natural can be even expanded to other forms of energy (like electrical energy) and non-natural building blocks. The chemically driven ATP synthesis, as in oxidative phosphorylation, represents a spontaneous process, in which the electrons of a fuel (glucose, NADH) are transferred to an electron acceptor such as oxygen with the concomitant generation of proton gradient, which is afterwards stored again as chemical energy in ATP. In the realm of synthetic biology, other options might also be feasible, for example: can we use electrical energy and directly plug it in to drive biological processes [4,5] or make use of natural electron transfer mechanisms to produce electricity? [6] The Electron Transport Chain-a Natural Toolbox of Functional Parts for the Construction of Artificial OrganellesDuring the process of oxidative phosphorylation, electrons are passed from an electron donor ("fuel") with a more negative One of the critical steps in sustaining life-mimicking processes in synthetic cells is energy, i.e., adenosine triphosphate (ATP) regeneration. Previous studies have shown that the simple addition of ATP or ATP regeneration systems, which do not regenerate ATP directly from ADP and P i , have no or only limited success due to accumulation of ATP hydrolysis products. In general, ATP regeneration can be achieved by converting light or chemical energy into ATP, which may also involve redox transformations of cofactors. The present contribution provides an overview of the existing ATP regeneration strategies and the related nicotinamide adenine dinucleotide (NAD + ) redox cycling, with a focus on compartmentalized systems. Special attention is being paid to those approach...

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.

Background The purpose of this work is to provide experimental evidence on the interactions of suspended nanoparticles with artificial or biological membranes and to assess the possibility of suspended nanoparticles interacting with the lipid component of biological membranes. Methods 1-Palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine (POPC) lipid vesicles and human red blood cells were incubated in suspensions of magnetic bare cobalt ferrite (CoFe 2 O 4 ) or citric acid (CA)-adsorbed CoFe 2 O 4 nanoparticles dispersed in phosphate-buffered saline and glucose solution. The stability of POPC giant unilamellar vesicles after incubation in the tested nanoparticle suspensions was assessed by phase-contrast light microscopy and analyzed with computer-aided imaging. Structural changes in the POPC multilamellar vesicles were assessed by small angle X-ray scattering, and the shape transformation of red blood cells after incubation in tested suspensions of nanoparticles was observed using scanning electron microscopy and sedimentation, agglutination, and hemolysis assays. Results Artificial lipid membranes were disturbed more by CA-adsorbed CoFe 2 O 4 nanoparticle suspensions than by bare CoFe 2 O 4 nanoparticle suspensions. CA-adsorbed CoFe 2 O 4 -CA nanoparticles caused more significant shape transformation in red blood cells than bare CoFe 2 O 4 nanoparticles. Conclusion Consistent with their smaller sized agglomerates, CA-adsorbed CoFe 2 O 4 nanoparticles demonstrate more pronounced effects on artificial and biological membranes. Larger agglomerates of nanoparticles were confirmed to be reactive against lipid membranes and thus not acceptable for use with red blood cells. This finding is significant with respect to the efficient and safe application of nanoparticles as medicinal agents.

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.

The bottom-up approach in synthetic biology aims to create molecular ensembles that reproduce the organization and functions of living organisms and strives to integrate them in a modular and hierarchical fashion toward the basic unit of life—the cell—and beyond. This young field stands on the shoulders of fundamental research in molecular biology and biochemistry, next to synthetic chemistry, and, augmented by an engineering framework, has seen tremendous progress in recent years thanks to multiple technological and scientific advancements. In this timely review of the research over the past decade, we focus on three essential features of living cells: the ability to self-reproduce via recursive cycles of growth and division, the harnessing of energy to drive cellular processes, and the assembly of metabolic pathways. In addition, we cover the increasing efforts to establish multicellular systems via different communication strategies and critically evaluate the potential applications.

The design of efficient schemes for nicotinamide adenine dinucleotide (NAD) regeneration is essential for the development of enzymatic biotechnological processes in order to sustain continuous production. In line with our motivation for the encapsulation of redox cascades in liposomes to serve as microbioreactors, we developed a straightforward strategy for the interfacial oxidation of entrapped NADH by ferricyanide as an external electron acceptor. Instead of the commonly applied enzymatic regeneration methods, we employed a hydrophobic redox shuttle embedded in the liposome bilayer. Tetracyanoquinodimethane (TCNQ) mediated electron transfer across the membrane and thus allowed us to shortcut and emulate part of the electron transfer chain functionality without the involvement of membrane proteins. To describe the experimental system, we developed a mathematical model which allowed for the determination of rate constants and exhibited handy predictive utility.