A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system

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 Escherichia coli crude extract. This new ATP regeneration system allows efficient recycling of inorganic phosphate, a strong inhibitor of protein synthesis. We show that up to 1.34–1.65 mg/mL of active reporter protein is synthesized in batch-mode reaction after 5 h of incubation. Unlike typical hybrid in vitro protein synthesis systems based on bacteriophage transcription, expression is carried out through E. coli 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.