Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System

Abstract

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

From vesicles toward protocells and minimal cells

“…189 In addition, Devaraj's group also succeeded in synthesising phospholipids using the enzyme, fatty acyl CoA ligase (FACL). 190 Similar membrane growth using an “anabolism type” of reaction scheme was reported by Toyota's group. 191 All these examples are highly challenging approaches that try to realize membrane growth systems that mimic contemporary cellular life systems.…”

“…Such vesicle-confined synthesis of membrane-forming molecules has already been achieved successfully by several groups. [180][181][182][183][184][185][186][187][188][189][190][191] However, these types of advanced methods are difficult to apply for the synthesis of large amounts of membrane molecules. Therefore, in many cases, bilayer membrane-forming molecules are delivered to the vesicles from the external bulk solution.…”

ATP Recycling Fuels Sustainable Glycerol 3-Phosphate Formation in Synthetic Cells Fed by Dynamic Dialysis

“…Synthetic biologists are joining forces internationally to build cellular mimics from the bottom up that stay away from thermodynamic equilibrium − and display life-like properties, including physicochemical homeostasis, − subcompartmentalization, − sensing/communication, − protein synthesis, , DNA replication, , energy and cofactor (re)­generation, ,,, membrane growth, − division, , and Darwinian evolution . Synthetic cells are envisioned as selectively open systems where phospholipid bilayers maintain electrochemical gradients and provide spatial confinement to a sustainable minimal metabolism, while embedded membrane proteins promote directional communication and transport with the external environment. ,,,, Importantly, internal recycling networks complementary to the protein-mediated exchange of nutrients and waste products are key to avoiding thermodynamic equilibrium.…”

In situ synthesis of artificial lipids

“…Taking into consideration the previous approach, Bhattacharya et al designed an efficient one-pot route to synthesize membrane-forming noncanonical phospholipids by repurposing the activity of a soluble ligase and using fatty acids as precursors (Figure 2a) [18]. In an initial enzymatic step, fatty acids were converted into the corresponding coenzyme A thioesters (FA-CoA) by FadD2, a mycobacterial fatty acyl-CoA ligase (FACL).…”