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.
Highlights
All living cells consist of membrane compartments, which are mainly composed of phospholipids
We synthesized dodecanoyl-AMP 117 (Supplementary Figs. 1–4) as a model fatty acyl adenylates (FAAs), and found that it was fairly stable to hydrolysis at 37 °C in 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, pH 7.5 in the absence or presence (10 mM) of Mg2+, and over time scales relevant to our subsequent experiments (Supplementary Fig. 5a)
Our strategy of using a soluble enzyme to promote phospholipid formation has a distinct advantage over previously described methods using integral membrane proteins[27,31,32]
Summary
All living cells consist of membrane compartments, which are mainly composed of phospholipids. Phospholipids are generated enzymatically by the reaction of a polar head group with long-chain acyl derivatives These key steps rely on integral membrane proteins, such as acyltransferases, which require preexisting membranes for proper folding and function[1] (Fig. 1a). Since present-day integral membrane proteins cannot carry out true de novo phospholipid formation, a method by which a soluble enzyme could facilitate the synthesis of membrane-forming phospholipids is required It can provide simplified strategies for generating membrane compartments in synthetic cells[6,7,8,9], enable the development of tools for reconstituting membrane proteins[10,11], and facilitate strategies for synthesizing structured lipids[12]. This demonstrates that pathways radically different from those taking place in living cells may be developed for synthesizing membrane-forming materials
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