The utilization efficiency of a secondary energy source in a cell-free protein synthesis system can be improved by use of a metabolic inhibitor. Oxalate, a potent inhibitor of phophoenolpyruvate synthetase, substantially increased the yield of chloramphenicol acetyltransferase synthesis through the enhanced supply of ATP. Oxalate, at 2.7 mM, increased the synthesis yield by 47% when successive amino acids additions prevent amino acid depletion during protein synthesis. These results suggest that cell-free protein synthesis efficiency could also be improved by disrupting the gene encoding phosphoenolpyruvate synthetase.