Difficulties associated with genetic manipulation and restrictions on the use of genetically modified organisms limit the application of metabolic engineering for improving the productivity of a cell. In this research, a system-oriented strategy was proposed to develop a regulatory defined medium (RDM) that removes the intracellular constraints of a cell system without any genetic manipulation. A transcriptional regulated metabolic model was first applied to identify the minimum secretion rate of the desired product and to recognize target genes that their up- or down-regulation enhances the lower bound of the production. Regulators of enzymes expressed by the target genes were extracted from the Brenda database and their effect on the production was experimentally evaluated. Then, design of experiment was applied to optimize the concentration of the selected compounds. The strategy was implemented on E. coli to improve ethanol production. Two inhibitors and four activators were selected for addition to the medium and their concentrations were optimized. The proposed RDM increased ethanol yield from 0.06 to 0.15 mol/mol carbon that demonstrates the effectiveness of the added compounds on the metabolism. This result indicates that the strategy was successful in reducing the intracellular constraints by adding regulatory compounds to the extracellular medium.