Compositions and methods are provided for the enhanced in vitro synthesis of polypeptides. In order to improve the performance of in vitro protein synthesis reactions, metabolic inhibitors, or manipulation of a source organism, is used to diminish or avoid the action of enzymes responsible for undesirable amino acids production or depletion. A homeostatic system may be used for production of ATP, where the required high energy phosphate bonds are generated in situ, e.g. through coupling with an oxidation reaction. The homeostatic energy source will typically lack high energy phosphate bonds itself, and will therefore utilize free phosphate in the reaction mix during generation of ATP. The homeostatic energy source is provided in combination with an enzyme that catalyzes the creation of high energy phosphate bonds and with an enzyme that can use that high energy phosphate bond to regenerate ATP.