{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T20:32:48Z","timestamp":1791664368611,"version":"4.3.4"},"reference-count":42,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microb Cell Fact"],"published-print":{"date-parts":[[2012,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by <jats:italic>Escherichia coli<\/jats:italic> in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in <jats:italic>E. coli<\/jats:italic> (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the <jats:italic>recA<\/jats:italic>, <jats:italic>deoR<\/jats:italic>, <jats:italic>nupG<\/jats:italic> and <jats:italic>endA<\/jats:italic> genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1\u2009kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100\u2009g\/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5\u03b1, carrying the same plasmid, was also cultivated under the same conditions.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (Y<jats:sub>P\/X<\/jats:sub>). The triple mutant VH33 \u0394 (<jats:italic>recA deoR nupG<\/jats:italic>) accumulated low amounts of acetate and resulted in the best Y<jats:sub>P\/X<\/jats:sub> (4.22\u2009mg\/g), whereas Y<jats:sub>P\/X<\/jats:sub> of strain VH33 only reached 1.16\u2009mg\/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186\u2009mg\/L of pDNA, 40\u2009g\/L of biomass and only 2.2\u2009g\/L of acetate. In contrast, DH5\u03b1 produced only 70\u2009mg\/L of pDNA and accumulated 9.5\u2009g\/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>The pDNA concentration obtained with the engineered strain VH33 \u0394 (<jats:italic>recA deoR nupG)<\/jats:italic> is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 \u0394 (<jats:italic>recA deoR nupG<\/jats:italic>) and its cultivation using elevated glucose concentrations represent an attractive technology for fast and efficient pDNA production and a valuable alternative to fed-batch cultivations of commercial strains.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1475-2859-11-132","type":"journal-article","created":{"date-parts":[[2012,9,19]],"date-time":"2012-09-19T20:20:57Z","timestamp":1348086057000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":56,"title":["Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode"],"prefix":"10.1186","volume":"11","author":[{"given":"Gheorghe M","family":"Borja","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eugenio","family":"Meza Mora","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Blanca","family":"Barr\u00f3n","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guillermo","family":"Gosset","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Octavio T","family":"Ram\u00edrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alvaro R","family":"Lara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2012,9,19]]},"reference":[{"key":"728_CR1","series-title":"Methods in Molecular Biology","doi-asserted-by":"publisher","first-page":"271","DOI":"10.1007\/978-1-61779-433-9_14","volume-title":"Recombinant Gene Expression","author":"AR Lara","year":"2012","unstructured":"Lara AR, Ram\u00edrez OT: Recombinant Gene Expression. 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