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Neal K Devaraj - One of the best experts on this subject based on the ideXlab platform.

  • expression of fatty acyl coa ligase drives one pot De Novo Synthesis of membrane bound vesicles in a cell free transcription translation system
    Journal of the American Chemical Society, 2021
    Co-Authors: Ahanjit Bhattacharya, Christy J Cho, Roberto J Brea, Neal K Devaraj
    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.

  • expression of fatty acyl coa ligase drives one pot De Novo Synthesis of membrane bound vesicles in a cell free transcription translation system
    bioRxiv, 2021
    Co-Authors: Ahanjit Bhattacharya, Christy J Cho, Roberto J Brea, Neal K Devaraj
    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.

Peter H. Seeberger - One of the best experts on this subject based on the ideXlab platform.

Rosalind A Coleman - One of the best experts on this subject based on the ideXlab platform.

  • triacsin c blocks De Novo Synthesis of glycerolipids and cholesterol esters but not recycling of fatty acid into phospholipid eviDence for functionally separate pools of acyl coa
    Biochemical Journal, 1997
    Co-Authors: R A Igal, P Wang, Rosalind A Coleman
    Abstract:

    The trafficking of acyl-CoAs within cells is poorly unDerstood. In orDer to Determine whether newly synthesized acyl-CoAs are equally available for the Synthesis of all glycerolipids and cholesterol esters, we incubated human fibroblasts with [14C]oleate, [3H]arachidonate or [3H]glycerol in the presence or absence of triacsin C, a fungal metabolite that is a competitive inhibitor of acyl-CoA synthetase. Triacsin C inhibited De Novo Synthesis from glycerol of triacylglycerol, diacylglycerol and cholesterol esters by more than 93%, and the Synthesis of phospholipid by 83%. However, the incorporation of oleate or arachidonate into phospholipids appeared to be relatively unimpaired when triacsin was present. Diacylglycerol acyltransferase and lysophosphatidylcholine acyltransferase had similar DepenDences on palmitoyl-CoA in both liver and fibroblasts; thus it did not appear that acyl-CoAs, when present at low concentrations, would be preferentially used to acylate lysophospholipids. We interpret these data to mean that, when fatty acid is not limiting, triacsin blocks the acylation of glycerol 3-phosphate and diacylglycerol, but not the reacylation of lysophospholipids. Two explanations are possible: (1) different acyl-CoA synthetases exist that vary in their sensitivity to triacsin; (2) an inDepenDent mechanism channels acyl-CoA towards phospholipid Synthesis when little acyl-CoA is available. In either case, the acyl-CoAs available to acylate cholesterol, glycerol 3-phosphate, lysophosphatidic acid and diacylglycerol and those acyl-CoAs that are used by lysophospholipid acyltransferases and by ceramiDe N-acyltransferase must resiDe in two non-mixing acyl-CoA pools or, when acyl-CoAs are limiting, they must be selectively channelled towards specific acyltransferase reactions.

Ahanjit Bhattacharya - One of the best experts on this subject based on the ideXlab platform.

  • expression of fatty acyl coa ligase drives one pot De Novo Synthesis of membrane bound vesicles in a cell free transcription translation system
    Journal of the American Chemical Society, 2021
    Co-Authors: Ahanjit Bhattacharya, Christy J Cho, Roberto J Brea, Neal K Devaraj
    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.

  • expression of fatty acyl coa ligase drives one pot De Novo Synthesis of membrane bound vesicles in a cell free transcription translation system
    bioRxiv, 2021
    Co-Authors: Ahanjit Bhattacharya, Christy J Cho, Roberto J Brea, Neal K Devaraj
    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.

Xuechen Li - One of the best experts on this subject based on the ideXlab platform.

  • total Synthesis of pseudomonas aeruginosa 1244 pilin glycan via De Novo Synthesis of pseudaminic acid
    Journal of the American Chemical Society, 2017
    Co-Authors: Yanfeng Zhang, Gloria Andolina, Xuechen Li
    Abstract:

    Pseudaminic acid (Pse) is a nonulosonic acid unique to bacterial species, found as a component of important cell surface glycans and glycoproteins in various pathogenic species, such as the critical hospital threat Pseudomonas aeruginosa. Herein we present the Development of a facile and scalable De Novo Synthesis of Pse and its functionalized Derivatives from easily available Cbz-l-allo-threonine methyl ester (16 steps in 11% yield). The key reactions in our De Novo Synthesis involve the diastereoselective glycine thioester isonitrile-based aldol-type reaction to create the 1,3-anti-diamino skeleton, followed by the Fukuyama reduction and the indium-mediated Barbier-type allylation. Moreover, we have studied the glycosylation of the Pse glycosyl donors and iDentified the structural Determinants for its glycosylation diastereoselectivity, which enabled us to complete the total Synthesis of P. aeruginosa 1244 pilin trisacchariDe α-5NβOHC47NFmPse-(2→4)-β-Xyl-(1→3)-FucNAc.