Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic byproduct that is synthesized aerobically when the glucose rate exceeds the limits. In recent years, several researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.