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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
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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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Abstract

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.

Peer review status:UNDER REVIEW

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