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Engineering E. coli for improved microaerobic pDNA production.

Karim E Jaén¹, Daniela Velazquez¹, Frank Delvigne²

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

Bioprocess and Biosystems Engineering

|May 13, 2019

Summary

This summary is machine-generated.

Engineering Escherichia coli strains by deleting the recombinase A (recA) gene and expressing Vitreoscilla hemoglobin (VHb) impacts plasmid DNA (pDNA) production. VHb improved pDNA yield in W3110 but not BL21 strains.

Keywords:

Oxygen limitation Plasmid DNA Redox sensor green Vitreoscilla hemoglobin

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Area of Science:

Biotechnology
Microbial Engineering
Molecular Biology

Background:

Escherichia coli is a key host for plasmid DNA (pDNA) production.
Optimizing pDNA yield requires understanding host physiology under various conditions.
Recombinase A (recA) and Vitreoscilla hemoglobin (VHb) are targets for strain improvement.

Purpose of the Study:

To engineer E. coli strains W3110 and BL21 for enhanced pDNA production.
To investigate the effects of recA deletion and VHb expression on pDNA yield and quality.
To evaluate strain performance under aerobic and oxygen-limited conditions.

Main Methods:

Genetic engineering of E. coli W3110 and BL21 strains.
Deletion of the recA gene and chromosomal insertion of the VHb gene (vgb).
Cultivation under aerobic conditions and shifts to microaerobic/oxygen-limited regimes.
Analysis of pDNA yield, supercoiled pDNA fraction (SCF), and metabolic by-products.

Main Results:

recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains.
VHb expression improved pDNA production in W3110 but not BL21.
VHb expression reduced fermentative by-products and increased oxidative activity.
Codon-optimized VHb in BL21 did not improve, but decreased, pDNA production.

Conclusions:

recA deletion is beneficial for pDNA quality (SCF).
VHb physiological effects differ between E. coli W3110 and BL21.
Strain selection and VHb optimization are crucial for efficient pDNA production.