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. 2010 Jun;4(2):85-93.
doi: 10.1007/s11693-009-9048-1. Epub 2009 Dec 3.

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

Giovanni Murtas  1
Affiliations

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

Giovanni Murtas. Syst Synth Biol. 2010 Jun.

Abstract

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.

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Figures

Fig. 1
Fig. 1
A liposome that builds its own membrane based on entrapped enzymes is the prototype of the simplest autopoietic minimal cell. P Precursors are the reagents required for an enzymatic catalysis, E enzyme/s controlling lipid catalysis, L lipids are released within the liposome lumen and spontaneously become incorporated into the lipid bilayer and this results in vesicle growth. The enzymatic pathway produces the palmitate represented as the dark shaded lipids
Fig. 2
Fig. 2
FAS-B catalysis and assay. a The Stoichiometry of the synthesis of palmitate controlled by the FAS-B enzyme from Brevibacterium ammoniagenes. The acetyl-CoA primes the reaction, while the elongation steps are sustained by malonyl-CoA. The free energy needed for the FAS functions is provided by the decarboxilation of the malonyl-CoA. Reducing power is provided by the oxidation of 14 molecules of NADPH for each molecule of palmitate. b FAS-B enzyme assay measuring the NADPH disappearance (oxidation) spectrophotometrically. The decline of absorbance of NADPH, when the FAS-B catalysis is active, (square) in comparison with the absorbance measured when malonyl-CoA was omitted, (circle). Each value reported in the graph equal the delta between the absorbance at 340 and 600 nm wavelength. The error bars reported in the graph represent the range of values of experiments performed in triplicate
Fig. 3
Fig. 3
Palmitate synthesis. Palmitate is synthesised in bulk solution or in liposomes, based on the FAS-B catalysis, and analysed by TLC. The palmitate released is 14C-labeled by [2-14C] malonyl-CoA incorporation. The following samples are separated by TLC after synthesis: line 1, palmitate synthesis (p.s.) entrapped in liposomes supplying Proteinase K externally (sample applied twice more concentrated); line 2, p.s. encapsulated in liposomes and no Proteinase K added externally; line 3, p.s. in liposomes without FAS-B enzyme; line 4, p.s. in liposomes without acetyl-CoA; line 5, p.s. in bulk solution and no Proteinase K supplied; line 6, p.s. in bulk solution with Proteinase K; line 7, [2-14C] malonyl-CoA (0.74 nmoles). The arrow shows the position expected for palmitate as determined in a separate TLC (same conditions) experiment with non-radioactive palmitic acid standard (not shown)
Fig. 4
Fig. 4
FRET assay for vesicle membrane growth. a Curve of normalised F don/F acc versus aliquots of palmitate micelles added on preformed POPC vesicles. b number weighted DLS size distribution of preformed vesicles re-extruded after re-hydration, the DLS analysis was performed before the incubation with palmitate micelles, c curve of normalised F don/F acc versus time (hours) as result of palmitate synthesis in the inner water pool of liposomes and incorporation in boundary layer. FRET assay for vesicle with internal FAS catalysis and Proteinase K added externally (diamonds), without precursor malonyl-CoA (squares), with no FAS-B enzyme in reaction (triangles), d number weighted DLS size distribution of vesicles re-extruded after re-hydration of the freeze-dried vesicles with the FAS-B catalysis solution, the DLS analysis was performed before the incubation period. The error bars reported in the graphs represent the range of values of experiments performed in triplicate
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