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Engineering E. coli for improved microaerobic pDNA production

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Abstract

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA−, which, together with W3110recA−vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

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Acknowledgements

Financial support from CONACyT Grant Number 256617 is acknowledged.

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Authors and Affiliations

  1. Posgrado en Ciencias Naturales e Ingeniería, Universidad Autónoma Metropolitana-Cuajimalpa, Vasco de Quiroga 4871, Santa Fe, 05348, Mexico City, Mexico

    Karim E. Jaén & Daniela Velazquez

  2. Gembloux Agro-Bio Tech, TERRA Research and Teaching Centre, Microbial Processes and Interactions (MiPI), University of Liege, Gembloux, Belgium

    Frank Delvigne

  3. Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa, Vasco de Quiroga 4871, Santa Fe, 05348, Mexico City, Mexico

    Juan-Carlos Sigala & Alvaro R. Lara

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  1. Karim E. Jaén
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  2. Daniela Velazquez
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  3. Frank Delvigne
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  4. Juan-Carlos Sigala
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  5. Alvaro R. Lara
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Correspondence to Alvaro R. Lara.

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Jaén, K.E., Velazquez, D., Delvigne, F. et al. Engineering E. coli for improved microaerobic pDNA production. Bioprocess Biosyst Eng 42, 1457–1466 (2019). https://doi.org/10.1007/s00449-019-02142-5

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  • DOI: https://doi.org/10.1007/s00449-019-02142-5

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