Therapeutic applications of plasmid DNA have significantly advanced during the last years and future demand for plasmid DNA requires the development of efficient bioprocesses. Concerning these facts, our aim is to optimize the yield of a therapeutic plasmid pcDNA3–FLAG–p53 using a new bacterial strain, Escherichia coli VH33. This innovative research combines the potential of VH33, which uses a different glucose transport allowing efficient growth and lower acetate production, for pDNA biosynthesis with a validated factorial design. This work achieved higher plasmid yields of 81.77 μg pDNA/mL, when compared with other results for similar assays which reached about 40 μg pDNA/mL. The strategy is based on the change of fermentation media composition in terms of nutrients, by the development of an experimental design directed to aromatic amino acids pathway. The results revealed which combination should be applied in terms of nitrogen and carbon source requirements in order to obtain 36.69 μg pDNA/mg cell dry mass of specific yields and 6.01% of purity on lysates. Overall, the proposal model show the influence of tyrosine, phenylalanine and tryptophan on their pathway, providing necessary precursors to nucleotides’ network, as well as temperature shift (37–42 °C) to increase the plasmid copy number per cell.