Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes
(a) Metabolic pathways that lead to the production of DPPG and DPPE starting from palmitoyl-CoA, glycerol-3-phosphate (G3P), cytidine triphosphate (CTP) and L-serine as main substrates. The two-step acyl transfer reaction and the first headgroup conversion step are common to the PE and PG pathways that then branch out into different headgroup modification reactions (see Supplementary text in S1 File for a description of the individual enzymatic steps). For the final step of PG synthesis there exist three alternative enzymes: PgpA, PgpB and PgpC, of which two (A/C) were used in this study. (b) Fluorescence scans of SDS-PAGE gels for the headgroup modifying enzymes produced in the PURE system. Fluorescently labeled lysine residues were incorporated during translation. The left gel is 15% polyacrylamide. In addition to the pssA gene product that was used as a control, the gene products of pgpA, pgpC and pgsA were synthesized. The right gel is 12% polyacrylamide and, besides the plsB gene product used as a control, the genes cdsA, pssA and psd were expressed. Size markers are in kDa. The arrowheads point to the observed protein molecular mass. The symbol “*” indicates the position of the band as expected from the nucleotide sequence of the genes (Supplementary text in S1 File). (c) Schematic of the inside-out proteoliposome reconstitution experiments and enzymatic cascade reactions, where all genes of a given pathway were expressed in PUREfrex and all specific substrates were supplied. (d) LC-MS data reporting lipid production in the PE and PG pathways under various experimental conditions. Combined gene expression and lipid biogenesis was carried out as illustrated in (c) using 25 ng of each linear DNA templates, 500 μM G3P, 100 μM palmitoyl-CoA, 1 mM CTP and 500 μM L-serine. Details of MS signatures for the different lipids are reported in Table A in S1 File. Lipids DPPE and DPPG were unambiguously detected in a pathway-specific manner. No PG is produced in the reconstituted PE pathway. Likewise, no PE was detected in the PG pathway. When the plsB gene is omitted the complete pathways are shut down. In the absence of the Psd enzyme, PE was not detected and its substrate lipid DPPS accumulated. Note that the MRM data for PS come from the MS optimizer results, not from separate experiments as used for the other compounds. Data are mean and s.e.m. of three independent experiments, except for the negative controls without plsB gene where two independent experiments were conducted. For each replicate the same sample was injected between one and four times in the MS, their averaged value was calculated and data are reported as the mean and standard error across the different trials.