UC San Diego

The bottom-up generation of lipid membranes from minimal precursors is a key objective in synthetic biology research. In living cells, the synthesis of lipid assemblies is ubiquitously coordinated by several membrane-bound enzymes. However, these pathways are tough to replicate, owing to the difficulty of reconstituting the enzymes in vitro. Lipid membrane synthesis from simple metabolic building blocks remains challenging. We demonstrate a chemoenzymatic schematic for lipid membrane generation, utilizing a bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA as precursors. Palmitoyl-CoA spontaneously reacts with a cysteine-modified lysophospholipid via native chemical ligation (NCL), generating noncanonical amidophospholipids that self-assemble into micron-sized membranes. The results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles. Utilizing this route, we further investigate the application of non-amphiphilic, minimal precursor pathway to generate lipid membranes. We explore the use of acetate as the carbon feedstock for the chemoenzymatic reaction strategy, generation palmitoyl-CoA in situ and coupling it with small molecule head group such as cysteine to form phospholipid analogs. Ubiquitous enzymes acetyl-CoA synthetase (ACS) from E. coli was used to generate acetyl-CoA from acetate and human acetyl-CoA carboxylase (ACC) was used to then synthesize malonyl-CoA from acetyl-CoA. Additionally, cgFAS catalyzed the in situ generation of palmitoyl-CoA from acetyl-CoA and malonyl-CoA. Finally, we explore the formation of a unique analog of sphingolipid membranes from water-soluble precursors that have markedly different biophysical properties to their natural counterparts. We show that numerous transition metal ions, particularly Cu(II), catalyze the selective O-acylation of the biologically occurring single-chain amphiphile sphingosylphosphorylcholine using fatty acyl phosphates or thioesters as acyl donors under mild aqueous conditions. This work demonstrates the value of de novo membrane generation strategies starting from minimal, water-soluble precursors. Utilizing such approaches, we can control both the chemical structures of the lipid analogs as well as their biophysical properties in aqueous media. This effort contributes towards understanding the fundamental requirements for bottom-up generation of lipid membranes, providing alternative strategies to those previously shown.