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Enhancing microaerobic plasmid DNA production by chromosomal expression of Vitreoscilla hemoglobin in E. coli
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  • Karim Jaén,
  • Daniela Velazquez,
  • Juan Sigala Alanis,
  • Alvaro Lara
Karim Jaén
Universidad Autónoma Metropolitana

Corresponding Author:[email protected]

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Daniela Velazquez
Universidad Autónoma Metropolitana
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Juan Sigala Alanis
Universidad Autónoma Metropolitana-Cuajimalpa
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Alvaro Lara
Universidad Autónoma Metropolitana
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Oxygen availability and overflow metabolism are often limiting factors in high cell-density cultures. In the present study, expression of Vitreoscilla hemoglobin in the chromosome of Escherichia coli was used as a strategy to improve plasmid DNA (pDNA) production in biphasic fed-batch cultures. During the fully aerobic batch phase, the strain expressing VHb accumulated 28 % less acetate and 19 % more pDNA than the non-expressing strain. The fed-batch phase was carried out with a change of regime from aerobic to microaerobic conditions. The pDNA yields from biomass increased consistently in the VHb-expressing strain during the whole culture, while decreased progressively for the non-expressing strain during microaerobic conditions. The ratio of positive/negative plasmid replication control molecules (RNAII/RNAI) were lower for the VHb-expressing than for the non-expressing strain. However, the final pDNA titer was ca. 74 % higher for the former. Flux balance analysis suggests that VHb presence increases the flux in anaplerotic pathways. The higher lactate production observed in VHb-expressing cells may be triggered by an increased demand of NAD+ in glycolysis under microaerobic conditions. These results are valuable for faster development of robust pDNA production processes.
09 Apr 2020Submitted to Biotechnology and Bioengineering
09 Apr 2020Assigned to Editor
09 Apr 2020Submission Checks Completed
15 Apr 2020Reviewer(s) Assigned
30 Apr 2020Review(s) Completed, Editorial Evaluation Pending