Herein, we report our results showing that the productivity of cell-free protein synthesis can be enhanced through the regulation of the in vitro metabolism of an energy source. In a reaction mixture utilizing 3-phosphoglycerate (3PG) as an energy source, the supply of ATP was significantly enhanced when the reaction mixture was supplied with sodium oxalate, a potent inhibitor of phosphoenolpyruvate synthetase (PPS). The productivity of protein synthesis was also increased by approximately 70% upon the addition of oxalate. It was presumed that this enhancement in ATP supply resulted from the prevention of the pyruvate -> PEP reaction, which causes nonproductive ATP consumption. For the initial presence of 2.1 mM sodium oxalate, approximately 720 mu g/ml chloramphenicol acetyltransferase (CAT) was produced after 3 h of incubation at 37 degrees C.