Growing Membranes In Vitro by Continuous Phospholipid Biosynthesis from Free Fatty Acids

Abstract

One of the key aspects that defines a cell as a living entity is its ability to self-reproduce. In this process, membrane biogenesis is an essential element. Here, we developed an in vitro phospholipid biosynthesis pathway based on a cascade of eight enzymes, starting from simple fatty acid building blocks and glycerol 3-phosphate. The reconstituted system yields multiple phospholipid species that vary in acyl-chain and polar headgroup compositions. Due to the high fidelity and versatility, complete conversion of the fatty acid substrates into multiple phospholipid species is achieved simultaneously, leading to membrane expansion as a first step toward a synthetic minimal cell.

Keywords: enzymatic conversion; membrane proteins; membranes; phospholipid biosynthesis; synthetic cell.

Figure 1

Schematic representation of the in vitro phospholipid biosynthesis pathway and protein purification. (a) FadD converts simple fatty acid (FA) building blocks into acyl-chain donors which are utilized by the enzymes PlsB and PlsC to form lysophosphatidic acid (LPA) and phosphatidic acid (PA), respectively. PA is further converted into CDP- diacylglycerol which serves as precursor for biosynthesis of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). (b) Coomassie stained SDS-PAGE gel of the eight lipid biosynthesis enzymes purified by Ni-NTA chromatography. The upper-band in lane 6 displays a dimer of PgpA. The two bands in lane 8 represent the two individual subunits of the Psd enzyme.

Figure 2

In vitro activity of purified FadD, PlsB and PlsC. (a) FadD activity: reactions were performed in the presence of purified FadD, as specified. Subsequently, 0.1% of the internal standard DDM was added. Products were analyzed by LC–MS, normalized for the internal standard and plotted on the y-axis. Hydropathy profile alignment of (b) E. coli PlsB and (c) PlsC (red line), with the averaged hydropathy profile of their bacterial protein family (black line). (d) PlsB and PlsC activity. Reactions were performed in the presence of purified enzyme and 0.1% DDM. Products were analyzed by LC–MS, normalized and plotted.

Figure 3

In vitro synthesis of PA by FadD, PlsB and PlsC. (a) Stepwise conversion of oleic acid into PA. Reactions were performed in the presence of purified enzymes reconstituted into liposomes. Products were analyzed by LC–MS, normalized for the internal standard POPG and plotted. (b) Synthesis of PA at high oleic acid concentration. Reactions were performed as described above, after which 0.1% of the internal standard DDM was added. Lipid products were analyzed as described above and plotted.

Figure 4

Synthesis of PA with varying acyl chain composition. An equimolar ratio of four different fatty acid species were mixed together and incubated with FadD, PlsB and with or without PlsC, and reconstituted into liposomes in the presence or absence (control) of ATP as indicated. Levels of the four fatty acid species (a), four LPA species (b) and nine PA species (c) are displayed for the three reaction conditions. Products were analyzed by LC–MS, normalized for the internal standard POPG and plotted.

Figure 5

In vitro biosynthesis of (a) PG, (b) PE and (c) mixtures of PE and PG from oleic acid and glycerol 3-phosphate. Reactions were performed in the presence of purified enzymes reconstituted into liposomes. Products were analyzed by LC–MS, normalized for the internal standard POPG and plotted.

Figure 6

Phospholipid biosynthesis induced membrane expansion of liposomes. Membrane expansion was measured with the R18-self-quenching assay. Fluorescent emission spectra of R18 containing liposomes excited at 540 nm were recorded for the liposomes before (trace 1) and after (trace 2) the addition of 2.7 mM oleic acid and the complete conversion of oleic acid into PA (trace 3) by FadD, PlsB and PlsC as described in the legends of Figure 3b. Addition of Triton X-100 results in maximum fluorescent levels (trace 4) and was used to normalize the samples.

Figure 7

Transmission electron microscopic imaging of in vitro synthesized membranes. (a) Different phases during phospholipid biosynthesis, starting with small unilamellar liposomes (a1), the addition of 1.5 mM oleic acid to yield large swollen vesicles (a2), conversion of oleic acid into phospholipid resulting in stacked bilayer formation (a3), which is reversed upon addition of EDTA (a4) to yield large liposomes. (b) Different phases during phospholipid biosynthesis upon the dosed addition of small amounts of oleic acid. Starting with small unilamellar vesicles (b1), first addition of 0.3 mM oleic acid which does not result in observable vesicle swelling (a2), and formation of large vesicles, after multiple additions of small amounts of oleic acid to a final concentration of 1.5 mM, converted into phospholipid (b3).