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bioRxiv · 10.1101/2021.04.22.440977

Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system

Bhattacharya, A.·Cho, C. J.·Brea, R. J.·Devaraj, N. K.
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Abstract

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their de novo formation from minimal chemical and biological elements. Here we describe a chemoenzymatic route to membrane-forming non-canonical 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 membrane formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding for the fatty acyl-CoA ligase with suitable lipid precursors, enabled spontaneous one-pot de novo synthesis of membrane-bound vesicles. Non-canonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin is co-expressed alongside the acyl CoA ligase, the in situ assembled membranes were spontaneously modified 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.

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Bhattacharya, A., Cho, C. J., Brea, R. J., Devaraj, N. K.. 2021-04-22. Expression of fatty acyl-CoA ligase drives one-pot de novo synthesis of membrane-bound vesicles in a cell free transcription-translation system. https://doi.org/10.1101/2021.04.22.440977

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