초록
<P><B>Abstract</B></P> <P>A new cost-effective metabolism providing an ATP-regeneration system for cell-free protein synthesis is presented. Hexametaphosphate, a polyphosphate molecule, is used as phosphate donor together with maltodextrin, a polysaccharide used as carbon source to stimulate glycolysis. Remarkably, addition of enzymes is not required for this metabolism, which is carried out by endogenous catalysts present in the <I>Escherichia coli</I> crude extract. This new ATP regeneration system allows efficient recycling of inorganic phosphate, a strong inhibitor of protein synthesis. We show that up to 1.34–1.65mg/mL of active reporter protein is synthesized in batch-mode reaction after 5h of incubation. Unlike typical hybrid <I>in vitro</I> protein synthesis systems based on bacteriophage transcription, expression is carried out through <I>E. coli</I> promoters using only the endogenous transcription–translation molecular machineries provided by the extract. We demonstrate that traditional expensive energy regeneration systems, such as creatine phosphate, phosphoenolpyruvate or phosphoglycerate, can be replaced by a cost-effective metabolic scheme suitable for cell-free protein synthesis applications. Our work also shows that cell-free systems are useful platforms for metabolic engineering.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Novel ATP-regeneration for cell-free protein synthesis. </LI> <LI> Hexametaphosphate as phosphate donor molecule. </LI> <LI> Glycolytic pathway activated by maltodextrin phosphorylation. </LI> <LI> Sustained ATP regeneration and iP recycling. </LI> <LI> 1.34–1.65mg/mL of active deGFP synthesized <I>in vitro</I>. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>