De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production
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...
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Veröffentlicht in: | Applied microbiology and biotechnology 2013, Vol.97 (2), p.611-620 |
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Sprache: | eng |
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Zusammenfassung: | 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 (MG1655
ΔendAΔrecAΔpgi
) produced 25-fold more pDNA (19.1 mg/g dry cell weight, DCW) than its parental strain, MG1655
ΔendAΔrecA
(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. |
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ISSN: | 0175-7598 1432-0614 |
DOI: | 10.1007/s00253-012-4308-5 |