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.