Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
(a) Schematic of vesicle-confined experiments. PUREfrex supplemented with the plsB and plsC genes and with 500 μM G3P was encapsulated inside liposomes using gentle rehydration of a lipid film covering sub-millimetre glass beads. Lipid composition consisted of DOPC, DOPE, DOPG, cardiolipin, TexasRed-DHPE and DSPE-PEG-biotin (Table B in S1 File). Swelling occurred at 4°C to avoid reaction initiation. Gene expression outside liposomes was inhibited by protein digestion. Lipid biosynthesis was triggered by external supply of 100 μM oleoyl-CoA (o-CoA). (b) Confocal microscopy images of liposomes after swelling. Vesicles were labelled with a membrane dye (Texas-Red). Scale bar is 5 μm. (c,d) Concentration of 18:1 LPA (c) and DOPA (d) synthesized in a one-pot reaction by GPAT and LPAAT enzymes produced outside liposomes composed of DOPG, DOPE and cardiolipin (Table B in S1 File). Lipid precursors were 500 μM G3P and 100 μM o-CoA (except in negative control). Error bars indicate s.e.m. of two injections of the same sample. (e,f) Concentration of 18:1 LPA (e) and DOPA (f) produced by GPAT and LPAAT enzymes generated inside liposomes. Three experimental configurations corresponding to different localizations of protein digestion were tested. As expected, addition of Proteinase K both inside and outside liposomes totally inhibited lipid synthesis. In the absence of Proteinase K 18:1 LPA and DOPA accumulated as a result of both internal and external acyltransferase production. Liposome-confined IVTT and lipid synthesis was demonstrated by supplementing Proteinase K outside vesicles according to the reaction scheme illustrated in (a). Data are mean and s.e.m. of three independent experiments. For each replicate the same sample was injected two times in the MS, their averaged value was calculated and data are reported as the mean and standard error across the three trials.