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. 2019 Jan 17;10(1):300.
doi: 10.1038/s41467-018-08174-x.

A minimal biochemical route towards de novo formation of synthetic phospholipid membranes

Ahanjit Bhattacharya  1 , Roberto J Brea  1 , Henrike Niederholtmeyer  1 , Neal K Devaraj  2
Affiliations

A minimal biochemical route towards de novo formation of synthetic phospholipid membranes

Ahanjit Bhattacharya et al. Nat Commun. .

Abstract

All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipid synthesis is catalyzed by membrane-bound enzymes, which themselves require pre-existing membranes for function. Thus, the principle of membrane continuity creates a paradox when considering how the first biochemical membrane-synthesis machinery arose and has hampered efforts to develop simplified pathways for membrane generation in synthetic cells. Here, we develop a high-yielding strategy for de novo formation and growth of phospholipid membranes by repurposing a soluble enzyme FadD10 to form fatty acyl adenylates that react with amine-functionalized lysolipids to form phospholipids. Continuous supply of fresh precursors needed for lipid synthesis enables the growth of vesicles encapsulating FadD10. Using a minimal transcription/translation system, phospholipid vesicles are generated de novo in the presence of DNA encoding FadD10. Our findings suggest that alternate chemistries can produce and maintain synthetic phospholipid membranes and provides a strategy for generating membrane-based materials.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
De novo formation of phospholipid membranes based on adenylate chemistry. a A representative phospholipid biosynthetic pathway (Kennedy pathway), which involves multiple membrane-bound enzyme-catalyzed steps, substrates and cofactors [GPAT, glycerol-3-phosphate acyl transferase; LPAAT, lysophosphatidic acid acyl transferase; G3P, glycerol 3-phosphate; CoA, coenzyme A; FACoA, fatty acyl coenzyme A; LPA, lysophosphatidic acid; PA, phosphatidic acid; PL, phospholipid]. b The proposed synthetic pathway of phospholipids, which involves a single soluble enzyme FadD10 and reactive lipid precursors [DDA, dodecanoic acid]. c De novo synthesis of phospholipids (3 or 5) by chemoselective reaction of the model FAA dodecanoyl-AMP (1) and amine-functionalized lysolipids (2 or 4). d Kinetics of phospholipid 3 (open black triangles, red line) formation by the reaction of FAA 1 with lysolipid 2 (open black circles, blue line). Integrated HPLC peak areas (205 nm) from three experiments were used to monitor the progress of the reaction. e HPLC/ELSD traces monitoring the selective formation of phospholipid 3 by reaction of FAA 1 and lysolipid 2 in the presence of 50 mM lysine
Fig. 2
Fig. 2
FadD10 mediated de novo formation and assembly of phospholipid membranes. a Time series of spinning disk confocal microscopy images depicting de novo phospholipid 3 vesicle formation resulting from the incubation of an aqueous solution of dodecanoic acid, lysolipid 2, ATP, MgCl2, FadD10, and 0.1 mol% Texas Red® DHPE at 37 °C. Scale bar: 10 µm. b Cryogenic-transmission electron microscopy (cryo-TEM) image of a de novo formed phospholipid vesicle showing the presence of membranes. Scale bar: 100 nm. c Kinetics of the consumption of lysolipid 2 (open black circles, blue line) and formation of phospholipid 3 (open black triangles, red line) at 37 °C. Integrated HPLC peak areas (205 nm) from three experiments were used to monitor the progress of the reaction. d Spinning disk confocal microscopy images of phospholipid 3 vesicles formed with Alexa Fluor® 488-labeled FadD10 and 0.1 mol% Texas Red® DHPE, showing association of the enzyme with the membrane. Scale bar: 10 µm
Fig. 3
Fig. 3
Membrane growth and division in a microfluidic device. a Schematic representation of a microfluidic chip utilized to entrap phospholipid 3 giant vesicles encapsulating Alexa Fluor® 488-labeled FadD10 and ATP. Reactive precursors (dodecanoic acid, lysolipid 4, ATP, and MgCl2) were continuously flowed with simultaneous spinning disk confocal microscopy. Scale bar: 50 µm. b Cryo-transmission electron microscopy (cryo-TEM) image of a large multilamellar vesicle prepared as per the vesicles used for the microfluidics experiments. Note the presence of abundant internal membranes. Scale bar: 100 nm. c Fluorescence microscopy images (Texas Red® channel) corresponding to vesicle growth over time. Scale bar: 10 µm. See Supplementary Movie 3. d Fluorescence microscopy images (Texas Red® channel) corresponding to vesicle division. The yellow arrows indicate the formation and departure of a daughter vesicle. Scale bar: 20 µm. Inset depicts the daughter vesicle in the Alexa Fluor® 488 channel. In c and d, the images are scaled logarithmically to enhance visibility of internal membranous structures. See Supplementary Movie 4. e HPLC-MS experiment demonstrating formation of phospholipid 5 (indicated in red arrow) upon addition of lysolipid 4 and other precursors to phospholipid 3 giant vesicles encapsulating FadD10
Fig. 4
Fig. 4
Linking gene expression of FadD10 to phospholipid synthesis. a Schematic representation of the cell-free expression of FadD10 and subsequent assembly of the de novo synthesized phospholipid into vesicles in the presence of appropriate reactive precursors [TX-TL: transcription/translation]. b SDS–PAGE analysis of the expression of FadD10 in the PURExpress® System. Lane L1: No DNA; Lane L2: DHFR DNA; Lane L3: FadD10 DNA. c HPLC/ELSD traces monitoring the formation of phospholipid 3 by incubation of PURExpress® System with an aqueous solution of dodecanoic acid, lysolipid 2, ATP and MgCl2 at 37 °C in the absence (gray line) or presence (orange line) of plasmid DNA coding for FadD10. d Spinning disk confocal microscopy of the in situ formed phospholipid vesicles in the PURExpress® System driven by FadD10 expression. Membranes were stained using 0.1 mol% Texas Red® DHPE dye. Scale bar: 5 µm. e Localization of sfGFP-FadD10 to the membrane of the vesicles formed upon addition of the plasmid encoding the former into PURE system. External proteins were digested by Proteinase K. Scale bar: 5 µm
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