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

2021 ◽  
Author(s):  
Ahanjit Bhattacharya ◽  
Christy J. Cho ◽  
Roberto J. Brea ◽  
Neal K. Devaraj
Keyword(s):  
De Novo ◽  
Fatty Acyl ◽  
Translation System ◽  
De Novo Synthesis ◽  
One Pot ◽  
Coa Ligase ◽  
A Cell ◽  
Membrane Bound

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

2021 ◽  
Author(s):  
Ahanjit Bhattacharya ◽  
Christy J. Cho ◽  
Roberto J. Brea ◽  
Neal K. Devaraj
Keyword(s):  
High Efficiency ◽  
De Novo ◽  
Fatty Acyl ◽  
Translation System ◽  
De Novo Synthesis ◽  
Dna Encoding ◽  
One Pot ◽  
Coa Ligase ◽  
A Cell ◽  
Membrane Bound

AbstractDespite 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.

De novo synthesis of phenolic dihydroxanthene near-infrared emitting fluorophores

2015 ◽  
Vol 13 (30) ◽  
pp. 8169-8172 ◽  
Author(s):  
Jean-Alexandre Richard
Keyword(s):  
Near Infrared ◽  
De Novo ◽  
De Novo Synthesis ◽  
One Pot

We report a flexiblede novosynthesis of phenolic dihydroxanthenes in 60–70% yield thanks to a one-pot cascade sequence.

A short de novo synthesis of nucleoside analogs

Science ◽  
2020 ◽  
Vol 369 (6504) ◽  
pp. 725-730 ◽  
Author(s):  
Michael Meanwell ◽  
Steven M. Silverman ◽  
Johannes Lehmann ◽  
Bharanishashank Adluri ◽  
Yang Wang ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Viral Infections ◽  
De Novo ◽  
Nucleoside Analogs ◽  
Aldol Reactions ◽  
Displacement Reaction ◽  
De Novo Synthesis ◽  
One Pot ◽  
Drug Discovery And Development ◽  
Locked Nucleic Acids

Nucleoside analogs are commonly used in the treatment of cancer and viral infections. Their syntheses benefit from decades of research but are often protracted, unamenable to diversification, and reliant on a limited pool of chiral carbohydrate starting materials. We present a process for rapidly constructing nucleoside analogs from simple achiral materials. Using only proline catalysis, heteroaryl-substituted acetaldehydes are fluorinated and then directly engaged in enantioselective aldol reactions in a one-pot reaction. A subsequent intramolecular fluoride displacement reaction provides a functionalized nucleoside analog. The versatility of this process is highlighted in multigram syntheses of d- or l-nucleoside analogs, locked nucleic acids, iminonucleosides, and C2′- and C4′-modified nucleoside analogs. This de novo synthesis creates opportunities for the preparation of diversity libraries and will support efforts in both drug discovery and development.

scholarly journals Sphingomyelinases and Liver Diseases

Biomolecules ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 1497
Author(s):  
Naroa Insausti-Urkia ◽  
Estel Solsona-Vilarrasa ◽  
Carmen Garcia-Ruiz ◽  
Jose C. Fernandez-Checa
Keyword(s):  
Signaling Pathways ◽  
Liver Diseases ◽  
De Novo ◽  
Biological Effects ◽  
Acyl Chain ◽  
Chemical Properties ◽  
Fatty Acyl ◽  
Fatty Acyl Chain ◽  
De Novo Synthesis ◽  
Novel Treatments

Sphingolipids (SLs) are critical components of membrane bilayers that play a crucial role in their physico-chemical properties. Ceramide is the prototype and most studied SL due to its role as a second messenger in the regulation of multiple signaling pathways and cellular processes. Ceramide is a heterogeneous lipid entity determined by the length of the fatty acyl chain linked to its carbon backbone sphingosine, which can be generated either by de novo synthesis from serine and palmitoyl-CoA in the endoplasmic reticulum or via sphingomyelin (SM) hydrolysis by sphingomyelinases (SMases). Unlike de novo synthesis, SMase-induced SM hydrolysis represents a rapid and transient mechanism of ceramide generation in specific intracellular sites that accounts for the diverse biological effects of ceramide. Several SMases have been described at the molecular level, which exhibit different pH requirements for activity: neutral, acid or alkaline. Among the SMases, the neutral (NSMase) and acid (ASMase) are the best characterized for their contribution to signaling pathways and role in diverse pathologies, including liver diseases. As part of a Special Issue (Phospholipases: From Structure to Biological Function), the present invited review summarizes the physiological functions of NSMase and ASMase and their role in chronic and metabolic liver diseases, of which the most relevant is nonalcoholic steatohepatitis and its progression to hepatocellular carcinoma, due to the association with the obesity and type 2 diabetes epidemic. A better understanding of the regulation and role of SMases in liver pathology may offer the opportunity for novel treatments of liver diseases.

scholarly journals Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

1983 ◽  
Vol 212 (1) ◽  
pp. 161-165 ◽  
Author(s):  
E M Danielsen ◽  
O Norén ◽  
H Sjöström
Keyword(s):  
Signal Sequence ◽  
Aminopeptidase N ◽  
Soluble Form ◽  
Translation System ◽  
Translation Product ◽  
Free Translation ◽  
Microsomal Membranes ◽  
A Cell ◽  
Membrane Bound ◽  
Small Intestinal

The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000. This was found to be sensitive to the action of endo-beta-N-acetylglucosaminidase H (EC 3.2.1.96), showing that aminopeptidase N undergoes transmembrane glycosylation during synthesis. The position of the signal sequence in aminopeptidase N was determined by a synchronized translation experiment. It was found that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence of microsomal fractions was found to be similar to that on one of the forms of the enzyme obtained from tunicamycin-treated organ-cultured intestinal explants.

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