Yield Production of Recombinant Plasmid DNA with Escherichia Coli in Fed-Batch Culture by Pseudo-Exponential Feeding

ABSTRACT

For preventing and treating debilitating diseases, plasmid DNA [pDNA] is emerging as the vector of choice for gene therapy and DNA vaccination. In this study, the development of a new implementation of control strategies for high-cell-density (HCD) culture fermentation of Escherichia coli was described to produce high level of cell mass for the mass production of bacterial plasmid. A yield of 2 g of pDNA/L was achieved by application of the newly developed techniques for HCD culture. We were also able to reduce the fermentation time for the highest production to only 3 h rather than 72 h. Furthermore, application of the HCD culture technique permits to keep the organism at a low growth rate to ensure high quality of the produced plasmid.

The kinetics of batch and fed-batch cultures of E. coli producing plasmid was investigated. The cell lag time, the maximum specific growth rate and (^Y=_D/U) were determined as 2 h., 1.7 h⁻¹ and 1.61 g·g⁻¹, respectively. In the fed-batch culture, different specific growth rates were set at 2.5 h⁻¹, 3.2 h⁻¹, and 4.5 h⁻¹ by pseudo-exponential feeding, and the expressions for the specific rate of substrate consumption, the growth kinetics and the product formation kinetics of each phase were obtained. The result shows that the concentrations of cell and plasmid can reach 93.0 g·L⁻¹ and 2.0 g·L⁻¹ respectively, and the quality of the DNA vaccine met the requirements for medical use.

Keywords:

• high-cell-density
• fed-batch
• DNA vaccine
• E. coli
• plasmid
• kinetics