Modified Escherichia coli B (BL21), a superior producer of plasmid DNA compared with Escherichia coli K (DH5α)

Biotechnology Core Laboratory, NIDDK NIH Bethesda, Bldg 14A Room 173, Maryland 20892, USA.
Biotechnology and Bioengineering (Impact Factor: 4.13). 11/2008; 101(4):831-6. DOI: 10.1002/bit.21973
Source: PubMed


Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.

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    • "Compared to the JM strains, E. coli BL21 which does not contain F' has many advantages such as faster cell growth, lower acetate accumulation and better glucose utilization, which is ascribed to more active sugar metabolism such as glyoxylate shunt, gluconeogenesis , anaplerotic pathways and TCA cycle. In addition, BL21 exhibits less sensitivity to metabolic stress resulted from producing a large amount of heterologous proteins (Phue et al., 2008; Son et al., 2011). "
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    ABSTRACT: 2'-Fucosyllactose (2-FL) is one of most abundant functional oligosaccharides in human milk, which is involved in many biological functions for human health. To date, most microbial systems for 2-FL production have been limited to use E. coli JM strains since they cannot metabolize lactose. In this study, E. coli BL21star(DE3) was engineered through deletion of the whole endogenous lactose operon and introduction of the modified lactose operon containing lacZ△M15 from E. coli K-12. Expression of genes for guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic enzymes and heterologous α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori allowed the engineered E. coli BL21star(DE3) to produce 2-FL with 3-times enhanced yield than the non-engineered E. coli BL21star(DE3). In addition, the titer and yield of 2-FL were further improved by adding the three aspartate molecules at the N-terminal of FucT2. Overall, 6.4g/L 2-FL with the yield of 0.225g 2-FL/g lactose was obtained in fed-batch fermentation of the engineered E. coli BL21star(DE3) expressing GDP-l-fucose biosynthetic enzymes and three aspartate tagged FucT2. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 07/2015; 210. DOI:10.1016/j.jbiotec.2015.06.431 · 2.87 Impact Factor
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    • "During the last decades, many strains of Escherichia coli such as DH5␣ [9], MG1655 [3], VH32 [10], have been created through a series of mutations to facilitate cloning of heterologous genes and maintain the stability of pDNA. The genetic modifications of VH33 strain led to a reduction of glucose uptake rate and consequently acetate synthesis. "
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    ABSTRACT: Therapeutic applications of plasmid DNA have significantly advanced during the last years and future demand for plasmid DNA requires the development of efficient bioprocesses. Concerning these facts, our aim is to optimize the yield of a therapeutic plasmid pcDNA3–FLAG–p53 using a new bacterial strain, Escherichia coli VH33. This innovative research combines the potential of VH33, which uses a different glucose transport allowing efficient growth and lower acetate production, for pDNA biosynthesis with a validated factorial design. This work achieved higher plasmid yields of 81.77 μg pDNA/mL, when compared with other results for similar assays which reached about 40 μg pDNA/mL. The strategy is based on the change of fermentation media composition in terms of nutrients, by the development of an experimental design directed to aromatic amino acids pathway. The results revealed which combination should be applied in terms of nitrogen and carbon source requirements in order to obtain 36.69 μg pDNA/mg cell dry mass of specific yields and 6.01% of purity on lysates. Overall, the proposal model show the influence of tyrosine, phenylalanine and tryptophan on their pathway, providing necessary precursors to nucleotides’ network, as well as temperature shift (37–42 °C) to increase the plasmid copy number per cell.
    Biochemical Engineering Journal 06/2015; 98. DOI:10.1016/j.bej.2015.02.001 · 2.47 Impact Factor
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    • "RecA has also a proteolytic activity that activates the Cop protein, a repressor of plasmid replication [27]. In general, it has been reported that recA mutants display higher stability [29] and often show a higher pDNA production than parental strains [11,22,30]. "
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    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. Conclusion 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.
    Microbial Cell Factories 09/2012; 11(1):132. DOI:10.1186/1475-2859-11-132 · 4.22 Impact Factor
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