A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

Abstract

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity--inside liposomes--of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within - which corresponds to the autopoietic growth mechanism.

Development of an artificial cell, from self-organization to computation and self-reproduction

“…This was observed without the addition of membrane protein insertion mechanisms suggesting that E. coli cell-free extracts contain a remaining strong activity for inner membrane protein insertion. With the PURE system, IMPs can be also expressed and integrated into phospholipid bilayers, although with a much lower efficiency (93). Despite many efforts, the development of an active membrane, using only the internal gene expression, has yet to be demonstrated.…”

Advances in Minimal Cell Models: a New Approach to Synthetic Biology and Origin of Life

“…The third case comes from our laboratory in Rome, see Fig. 7A (Kuruma et al 2009). This work was inspired by a previous study done on lipid-synthesizing enzymes inside vesicles, at the aim of creating a lipid-producing liposome (Schmidli et al, 1991).…”

“…However the efficient production of PC was observed only in the case of short chain acyls, and the yield was around 10%. In the work of Kuruma et al (2009) In turn, the newly formed T7 RNA polymerase transcribes the mRNA of GFP, which is then translated to give the fluorescent protein (Ishikawa et al, 2004). (B) -Hemolysin, produced inside a giant vesicle from its DNA sequence, self-assembles into an heptamer which is a pore on the phospholipid membrane.…”

“…The two proteins, which -on the basis of their solubility -should be an integral membrane protein and a membrane associated one, respectively, catalyse the formation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA) from glycerol-3-phosphate (G3P) and palmitoyl-and oleoyl-coenzimeA (C16CoA, and C18:1CoA, respectively), through the intermediate lysophosphatidic acid (LPA). Notice that the membrane composition is a key factor for realizing a functional system (POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine; POPE: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine; POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol; CL: cardiolipin) (Kuruma et al, 2009). (B) Starting from Q-replicase-codifying (+)RNA strand and cell-free protein expression system, the production of Q-replicase was carried out inside liposomes, so that the complementary (-)RNA strand is produced from nucleotides and (+)RNA template.…”

DNA Crown Cells: Synthesis and Self-replication

“…Artificial cells, including liposomes, nanoparticles, and microcapsules, have been studied since the 1960s and have led to impressive achievements in the generation of artificial cells (Zhang et al, 2008;Lin et al 2013; Kuruma et al 2009). However, to date, artificial cells which can replicate autonomously have not been reported.…”