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Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing.
J Biotechnol. 2014 Sep 30; 186:119-27.JB

Abstract

The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L.

Authors+Show Affiliations

MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.MIT-Portugal Program, Portugal; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, United States.MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.Nature Technology Corporation, Lincoln, NE, United States.MIT-Portugal Program, Portugal; IBB - Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal. Electronic address: miguelprazeres@ist.utl.pt.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24995846

Citation

Gonçalves, Geisa A L., et al. "Plasmid DNA Production With Escherichia Coli GALG20, a Pgi-gene Knockout Strain: Fermentation Strategies and Impact On Downstream Processing." Journal of Biotechnology, vol. 186, 2014, pp. 119-27.
Gonçalves GA, Prather KL, Monteiro GA, et al. Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing. J Biotechnol. 2014;186:119-27.
Gonçalves, G. A., Prather, K. L., Monteiro, G. A., Carnes, A. E., & Prazeres, D. M. (2014). Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing. Journal of Biotechnology, 186, 119-27. https://doi.org/10.1016/j.jbiotec.2014.06.008
Gonçalves GA, et al. Plasmid DNA Production With Escherichia Coli GALG20, a Pgi-gene Knockout Strain: Fermentation Strategies and Impact On Downstream Processing. J Biotechnol. 2014 Sep 30;186:119-27. PubMed PMID: 24995846.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: fermentation strategies and impact on downstream processing. AU - Gonçalves,Geisa A L, AU - Prather,Kristala L J, AU - Monteiro,Gabriel A, AU - Carnes,Aaron E, AU - Prazeres,Duarte M F, Y1 - 2014/07/01/ PY - 2014/04/01/received PY - 2014/06/04/revised PY - 2014/06/04/accepted PY - 2014/7/5/entrez PY - 2014/7/6/pubmed PY - 2015/6/13/medline KW - DNA vaccine KW - Escherichia coli KW - Fermentation KW - Plasmid biopharmaceutical SP - 119 EP - 27 JF - Journal of biotechnology JO - J. Biotechnol. VL - 186 N2 - The market development of plasmid biopharmaceuticals for gene therapy and DNA vaccination applications is critically dependent on the availability of cost-effective manufacturing processes capable of delivering large amounts of high-quality plasmid DNA (pDNA) for clinical trials and commercialization. The producer host strain used in these processes must be designed to meet the upstream and downstream processing challenges characteristic of large scale pDNA production. The goal of the present study was to investigate the effect of different glucose feeding strategies (batch and fed-batch) on the pDNA productivity of GALG20, a pgi Escherichia coli strain potentially useful in industrial fermentations, which uses the pentose phosphate pathway (PPP) as the main route for glucose metabolism. The parental strain, MG1655ΔendAΔrecA, and the common laboratory strain, DH5α, were used for comparison purposes and pVAX1GFP, a ColE1-type plasmid, was tested as a model. GALG20 produced 3-fold more pDNA (∼141 mg/L) than MG1655ΔendAΔrecA (∼48 mg/L) and DH5α (∼40 mg/L) in glucose-based fed-batch fermentations. The amount of pDNA in lysates obtained from these cells was also larger for GALG20 (41%) when compared with MG1655ΔendAΔrecA (31%) and DH5α (26%). However, the final quality of pDNA preparations obtained with a process that explores precipitation, hydrophobic interaction chromatography and size exclusion was not significantly affected by strain genotype. Finally, high cell density fed-batch cultures were performed with GALG20, this time using another ColE1-type plasmid, NTC7482-41H-HA, in pre-industrial facilities using glucose and glycerol. These experiments demonstrated the ability of GALG20 to produce high pDNA yields of the order of 2100-2200 mg/L. SN - 1873-4863 UR - https://www.unboundmedicine.com/medline/citation/24995846/Plasmid_DNA_production_with_Escherichia_coli_GALG20_a_pgi_gene_knockout_strain:_fermentation_strategies_and_impact_on_downstream_processing_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0168-1656(14)00289-2 DB - PRIME DP - Unbound Medicine ER -