De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production
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The interest in plasmid DNA (pDNA) as a biopharmaceutical has been increasing over the last several years, especially after the approval of the first DNA vaccines. New pDNA production strains have been created by rationally mutating genes selected on the basis of Escherichia coli central metabolism and plasmid properties. Nevertheless, the highly mutagenized genetic background of the strains used makes it difficult to ascertain the exact impact of those mutations. To explore the effect of strain genetic background, we investigated single and double knockouts of two genes, pykF and pykA, which were known to enhance pDNA synthesis in two different E. coli strains: MG1655 (wild-type genetic background) and DH5α (highly mutagenized genetic background). The knockouts were only effective in the wild-type strain MG1655, demonstrating the relevance of strain genetic background and the importance of designing new strains specifically for pDNA production. Based on the obtained results, we created a new pDNA production strain starting from MG1655 by knocking out the pgi gene in order to redirect carbon flux to the pentose phosphate pathway, enhance nucleotide synthesis, and, consequently, increase pDNA production. GALG20 (MG1655ΔendAΔrecAΔpgi) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655ΔendAΔrecA (0.8 mg/g DCW), in glucose. For the first time, pgi was identified as an important target for constructing a high-yielding pDNA production strain.
KeywordsDNA vaccine Plasmid biopharmaceuticals Escherichia coli Strain engineering Metabolic engineering
This work was supported by the MIT-Portugal Program and Fundação para a Ciência e a Tecnologia (project PTDC/EBB-EBI/113650/2009 PhD grant SFRH/BD/33786/2009 to Geisa A. L. Gonçalves). We also acknowledge Kevin Solomon (MIT) for providing the plasmid pKD46recA+, Sang-Hwal Yoon (MIT) for constructing MG1655ΔendAΔrecA, and Diana Bower (MIT) for the development of HPLC methods. Special acknowledgment to all members of the Prather Research Group for providing helpful insights that contributed to this work.
Conflict of interest
The authors have declared no conflict of interest.
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