title: Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes.
abstract: The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes. Acyl-coenzyme A and glycerol-3-phosphate were used as canonical precursors to generate a variety of important bacterial lipids. Moreover, this study demonstrates that two-step acyl transfer can occur from enzymes synthesized inside vesicles. Besides clear implications for growth and potentially division of a synthetic cell, we postulate that gene-based lipid biosynthesis can become instrumental for ex vivo and protein purification-free production of natural and non-natural lipids.
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Andrew
authors.initials: A
authors.lastname: Scott
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Marek J
authors.initials: MJ
authors.lastname: Noga
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Paul
authors.initials: P
authors.lastname: de Graaf
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Ilja
authors.initials: I
authors.lastname: Westerlaken
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Esengul
authors.initials: E
authors.lastname: Yildirim
authors.affiliation: Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ, Delft, The Netherlands.
authors.forename: Christophe
authors.initials: C
authors.lastname: Danelon
mesh_terms: D000217:Acyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D002462:Cell Membrane / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D006706:Homeostasis; D008081:Liposomes / Q000378:metabolism*; D010743:Phospholipids / Q000096:biosynthesis*; D058615:Synthetic Biology
chemical_list: D008081:Liposomes; D010743:Phospholipids; D004295:Dihydroxyphenylalanine; D000217:Acyltransferases; D005992:Glycerol-3-Phosphate O-Acyltransferase; C018764:2-acylglycerophosphate acyltransferase