title: Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode.
abstract: BACKGROUND
  Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli 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 E. coli (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 recA, deoR, nupG and endA 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 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions.
  
  
  RESULTS
  The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation.
  
  
  CONCLUSIONS
  The pDNA concentration obtained with the engineered strain VH33 Δ (recA deoR nupG) is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 Δ (recA deoR nupG) 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.
authors.affiliation: Departamento de Medicina Molecular y Bioprocesos, Col. Chamilpa, CP 62210, Cuernavaca, Morelos, Mexico.
authors.forename: Gheorghe M
authors.initials: GM
authors.lastname: Borja
authors.forename: Eugenio
authors.initials: E
authors.lastname: Meza Mora
authors.forename: Blanca
authors.initials: B
authors.lastname: Barrón
authors.forename: Guillermo
authors.initials: G
authors.lastname: Gosset
authors.forename: Octavio T
authors.initials: OT
authors.lastname: Ramírez
authors.forename: Alvaro R
authors.initials: AR
authors.lastname: Lara
mesh_terms: D000941:Antigens / Q000235:genetics / Q000378:metabolism; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics; D009109:Mumps virus / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011938:Rec A Recombinases / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D019444:Vaccines, DNA / Q000096:biosynthesis
chemical_list: D000941:Antigens; C489070:DeoR protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C098551:NupG protein, E coli; D012097:Repressor Proteins; D019444:Vaccines, DNA; D011938:Rec A Recombinases; D005947:Glucose