Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: Fermentation strategies and impact on downstream processing

Gonçalves, Geisa A. L.; Prather, Kristala L. J.; Monteiro, Gabriel A.; Carnes, Aaron E.; Prazeres, D.M.F.

doi:10.1016/j.jbiotec.2014.06.008

Cited by 24 publications

(23 citation statements)

References 50 publications

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“…In order to attempt to isolate the effect of the operating mode, the plasmid productivity reported in batch, fed‐batch and perfusion cultures in bench scale bioreactors using E. coli DH5α as a host strain is presented in Table . In this work, a plasmid productivity of 3.00 ± 0.65 mg L −1 h −1 was obtained using a defined medium, which represents a 1.5‐fold increase in productivity when compared with a recent result for a bench fed‐batch process using a complex media and E. coli DH5α to produce a plasmid of 4 kbp …”

Section: Resultsmentioning

confidence: 55%

“…While in the batch phase the growth rate is not controlled (μ = μ m ), in the EFP phase the growth rate can be set to a specific value (μ < μ m ) by manipulating the inlet feed rate. The control of growth rate during pDNA production can result in high pDNA yield and high quality of the final product …”

Section: Methodsmentioning

confidence: 99%

“…Performance of industrial microorganisms as cell factories usually requires careful manipulation of process conditions, or strain improvement . The establishment of a high yield and high quality pDNA production process can be affected by growth rate, plasmid size and other cultivation conditions …”

Section: Introductionmentioning

confidence: 99%

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Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

García-Rendón

Munguía-Soto

Montesinos-Cisneros

et al. 2016

J of Chemical Tech & Biotech

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BACKGROUND The clinical application of plasmid DNA (pDNA) vaccines is progressing and it is essential to develop industrial bioprocesses to economically manufacture these macromolecules. In order to contribute to achieving this goal, in this work an exponential‐fed perfusion (EFP) culture for the production of pVAX1‐NH36 hosted in Escherichia coli DH5α for application as specific vaccine was investigated using a theoretical–experimental approach. RESULTS An experimental plasmid productivity of 3.0 mg L−1 h−1 was obtained, which represent a 1.5‐fold increase in this performance criteria. A mathematical model to investigate the system performance was built and validated. A novel aspect of the study is the analysis of the influence of individual model parameters on selected output variables and on scale‐up. CONCLUSIONS This work contributes to the design and optimization of EFP cultures for the efficient production of pDNA for therapeutic use. The higher productivity of the EFP bioprocess might reduce both capital investment and operational costs and as a result be economically attractive compared with conventional bioprocess. © 2016 Society of Chemical Industry

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Section: Resultsmentioning

confidence: 55%

Section: Methodsmentioning

confidence: 99%

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Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

García-Rendón

Munguía-Soto

Montesinos-Cisneros

et al. 2016

J of Chemical Tech & Biotech

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“…For example, the best volumetric productivities of minicircles obtained during the cell culture step reported in the literature are still only of the order of 0.5-9.0 mg/L (Kreiss et al 1998;Chen et al 2005;Mayrhofer et al 2008;Kay et al 2010;Gaspar et al 2014). This offers a striking contrast with the developments observed in plasmid manufacturing, which have pushed fermentation productivities up to 2.2-2.6 g/L (Carnes et al 2011;Gonçalves et al 2014). The low performance of the fermentation processes used to produce minicircle could be ascribed to (i) low expression of recombinases (Bigger et al 2001;Jechlinger et al 2004), (ii) low recombinase activity in cells reaching stationary phase (Kay et al 2010), and (iii) susceptibility of recombinases to changes in pH, temperature, and metabolite accumulation (Chen et al 2005;Simcikova et al 2014).…”

Section: Introductionmentioning

confidence: 94%

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

Šimčíková

Alves

Brito

et al. 2016

Appl Microbiol Biotechnol

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“…Accordingly, it is necessary to gain knowledge of the physiological responses of E. coli (the host for pDNA production) that may be triggered under large‐scale culture conditions and its impact on pDNA production. Several published reports have focused on the development of better production strains (Borja et al, ; Gonçalves et al, , ), culture media (Carnes et al, , Ongkudon et al, ), and cultivation schemes (Gonçalves et al, ; Jaén et al, ) to improve pDNA production.…”

Section: Introductionmentioning

confidence: 99%

Physiological effects of pH gradients on Escherichia coli during plasmid DNA production

Cortés¹,

Flores

Bolívar

et al. 2015

Biotech & Bioengineering

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A two-compartment scale-down system was used to mimic pH heterogeneities that can occur in large-scale bioreactors. The system consisted of two interconnected stirred tank reactors (STRs) where one of them represented the conditions of the bulk of the fluid and the second one the zone of alkali addition for pH control. The working volumes ratio of the STRs was set to 20:1 in order to simulate the relative sizes of the bulk and alkali addition zones, respectively, in large-scale bioreactors. Residence times (tR ) in the alkali addition STR of 60, 120, 180, and 240 s were simulated during batch cultures of an engineered Escherichia coli strain that produced plasmid DNA (pDNA). pH gradients of up to 0.9 units, between the two compartments, were attained. The kinetic, stoichiometric, and pDNA topological changes due to the pH gradients were studied and compared to cultures at constant pH of 7.2 and 8.0. As the tR increased, the pDNA and biomass yields, as well as pDNA final titer decreased, whereas the accumulation of organic acids increased. Furthermore, the transcriptional response of 10 selected genes to alkaline stress (pH 8.0) and pH gradients was monitored at different stages of the cultures. The selected genes coded for ion transporters, amino acids catabolism enzymes, and transcriptional regulators. The transcriptional response of genes coding for amino acids catabolism, in terms of relative transcription level and stage of maximal expression, was different when the alkaline stress was constant or transient. This suggests the activation of different mechanisms by E. coli to cope with pH fluctuations compared to constant alkaline pH. Moreover, the transcriptional response of genes related to negative control of DNA synthesis did not correlate with the lower pDNA yields. This is the first study that reports the effects of pH gradients on pDNA production by E. coli cultures. The information presented can be useful for the design of better bioreactor scale-up strategies.

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Plasmid DNA production with Escherichia coli GALG20, a pgi-gene knockout strain: Fermentation strategies and impact on downstream processing

Cited by 24 publications

References 50 publications

Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

Performance analysis of exponential‐fed perfusion cultures for pDNA vaccines production

Improvement of DNA minicircle production by optimization of the secondary structure of the 5′-UTR of ParA resolvase

Physiological effects of pH gradients on Escherichia coli during plasmid DNA production

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