Plasmid DNA (pDNA) is a vital product for the pharmaceutical industry, which can be injected directly in the form of DNA vaccines, and its significance and demand are increasing. The DNA vaccines require high production of the pDNA in form of supercoiled plasmid DNA (sc-pDNA) for their actual development. Several factors, including the host strain, the type of plasmid, and production process, influence the production and productivity of pDNA. In this study, the rnaII gene, necessary for ColE I plasmid replication initiation, along with the nrdAB genes involved in the dNTP’s synthesis pathway, were integrated into the Escherichia coli ( E. coli) genome. This integration effectively improved plasmid production by enhancing the replication initiation and increasing the supply of replication substrates. Additionally, a rich fed fermentation medium was used, and the fermentation process was optimized. After an initial period of fermentation, a gradient-fed strategy was implemented to control specific growth rates. As a result, the fermentation broth reached an OD ₆₀₀ of 113.7, with a plasmid production of 2.77 g/L and a supercoiled proportion of 97.3%.