Toward the assembly of a minimal divisome

Abstract

The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.

Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes

“…Besides volume expansion, we postulate that in vesiculo lipid biosynthesis could be exploited to change the equilibrium state of the membrane and triggers asymmetric division [ 41 ]. First, in light of the recently unveiled mechanism of L form cell reproduction [ 42 ], we predict that internal synthesis of phospholipids could be sufficient to induce shape deformation as a manifestation of the excess surface area of the membrane.…”

Self-replication of DNA by its encoded proteins in liposome-based synthetic cells

“…The in vitro construction of a minimal cell from separate biochemical parts (existing or novel) forms one of the outstanding challenges in synthetic biology 1 – 9 . Unlike abiogenic approaches to develop protocells that solely rely on compounds that were plausibly present on the primordial Earth 7 , 10 – 12 , the biosynthetic framework exploits the compositional diversity of the modern biology to assemble a minimal—yet living—cell.…”

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

“…Notable achievements include the reconstitution of the minimal translation machinery from Escherichia coli 1 , the yeast DNA replication apparatus 2 , filopodial structures 3 , cytoskeleton self-organization and centrosome positioning 4 , egg cytokinesis signaling 5 , DNA segregation with Par 6 , and clathrincoated buds 7 . Encouraged by the many cellular pieces that have already been reconstituted in vitro, synthetic biologists have now engaged in the construction of an entire cell [8][9][10][11][12] .…”