Here, we introduce a one-pot method for the bottom-up assembly of complex single- and multicompartment synthetic cells. Cellular components are enclosed within giant unilamellar vesicles (GUVs), produced at the milliliter scale directly from small unilamellar vesicles (SUVs) or proteoliposomes with only basic laboratory equipment within minutes. Toward this end, we layer an aqueous solution, containing SUVs and all biocomponents, on top of an oil–surfactant mix. Manual shaking induces the spontaneous formation of surfactant-stabilized water-in-oil droplets with a spherical supported lipid bilayer at their periphery. Finally, to release GUV-based synthetic cells from the oil and the surfactant shell into the physiological environment, we add an aqueous buffer and a droplet-destabilizing agent. We prove that the obtained GUVs are unilamellar by reconstituting the pore-forming membrane protein α-hemolysin and assess the membrane quality with cryotransmission electron microscopy (cryoTEM), fluorescence recovery after photobleaching (FRAP), and zeta-potential measurements as well as confocal fluorescence imaging. We further demonstrate that our GUV formation method overcomes key challenges of standard techniques, offering high volumes, a flexible choice of lipid compositions and buffer conditions, straightforward coreconstitution of proteins, and a high encapsulation efficiency of biomolecules and even large cargo including cells. We thereby provide a simple, robust, and broadly applicable strategy to mass-produce complex multicomponent GUVs for high-throughput testing in synthetic biology and biomedicine, which can directly be implemented in laboratories around the world.

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N 2 , the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N 2 H 4 ). Previously, we identified N 2 -producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one of 10 paralogs related to octaheme hydroxylamine (NH 2 OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAO-related hydroxylamine-oxidizing enzyme kustc1061 from K. stuttgartiensis. Interestingly, the HDH trimers formed octamers in solution, each octamer harboring an amazing 192 c-type heme moieties. Whereas HAO and kustc1061 are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well defined HAO and kustc1061. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (P 460 ) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N 2 -producing enzyme, next to nitrous oxide (N 2 O) reductase from denitrifying microorganisms.An estimated 30 -70% of all N 2 that is released into the atmosphere is produced by anaerobic ammonium-oxidizing (anammox) 4 bacteria (1, 2), which represent one of the latest scientific discoveries in the biogeochemical nitrogen cycle. These organisms gain their energy for growth from the oxidation of ammonium, with nitrite as the electron acceptor, to produce N 2 . With the employment of advanced molecular tools, these bacteria have been detected in nearly every anoxic environment where fixed nitrogen compounds are degraded (3). Besides its biogeochemical and ecological relevance, the anammox process has found worldwide application in ammonium removal from wastewater as an environment-friendly and cost-effective alternative to conventional systems (4).In our current understanding, anammox catabolism is composed of three consecutive, coupled reactions with two intermediates, nitric oxide (NO) and hydrazine (N 2 H 4 ): 1) the oneelectron reduction of the substrate nitrite to NO (Reaction 1); 2) the activation of the second substrate ammonium with NO and the concomitant input of three electrons to synthesize N 2 H 4 (Reaction 2); and 3) the oxidation of hydrazine, the most powerful reductant in nature, to N 2 (Reaction 3) (5-7). NO

Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (−750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.

Cytoskeletal elements, like actin and myosin, have been reconstituted inside lipid vesicles toward the vision to reconstruct cells from the bottom up. Here, we realize the de novo assembly of entirely artificial DNA-based cytoskeletons with programmed multifunctionality inside synthetic cells. Giant unilamellar lipid vesicles (GUVs) serve as cell-like compartments, in which the DNA cytoskeletons are repeatedly and reversibly assembled and disassembled with light using the cis – trans isomerization of an azobenzene moiety positioned in the DNA tiles. Importantly, we induced ordered bundling of hundreds of DNA filaments into more rigid structures with molecular crowders. We quantify and tune the persistence length of the bundled filaments to achieve the formation of ring-like cortical structures inside GUVs, resembling actin rings that form during cell division. Additionally, we show that DNA filaments can be programmably linked to the compartment periphery using cholesterol-tagged DNA as a linker. The linker concentration determines the degree of the cortex-like network formation, and we demonstrate that the DNA cortex-like network can deform GUVs from within. All in all, this showcases the potential of DNA nanotechnology to mimic the diverse functions of a cytoskeleton in synthetic cells.

SignificanceVirophages are parasites of giant viruses within protists. They reduce giant virus production and increase host cell survival. They provide a defense system for protists against giant viruses in diverse environments, likely with ecological relevance for protist populations. To understand the remarkable virophage life cycle, it is crucial to investigate how they assemble into infectious particles and which processes require interactions with giant virus and host. We examined the marine virophage mavirus to show that its major and minor capsid proteins assemble into virus-like particles in the absence of specific host or viral factors. Subsequently, the virophage-encoded protease processes the major capsid protein to prepare virions for infection.