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.
    Full-text · Article · Jul 2015 · Journal of Biotechnology
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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.
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    • "New adjuvants (Lurescia et al., 2014; Capitani et al., 2014) Nomenclature p number of parameters N number of data points X biomass concentration S glucose concentration G glycerol concentration A acetate concentration P plasmid concentration r X rate of biomass production r S rate of glucose consumption r G rate of glycerol consumption r A rate of acetate consumption r PA rate of acetate production r P rate of plasmid production T total specific growth rate S specific growth rate on glucose G specific growth rate on glycerol A specific growth rate on acetate maxS maximum specific growth rate on glucose maxG maximum specific growth rate on glycerol maxA maximum specific growth rate on acetate K S affinity constant for glucose K G affinity constant for glycerol K A affinity constant for acetate Ki G−S inhibition constant of growth on glycerol by glucose Ki A−S inhibition constant of growth on acetate by glucose Ki A−G inhibition constant of growth on acetate by glycerol Ki S−A inhibition constant of growth on glucose by acetate Ki G−A inhibition constant of growth on glycerol by acetate Ki X−A inhibition constant of biomass growth by acetate Ki X−P inhibition constant of biomass growth by plasmid production Ki P−A inhibition constant of plasmid production by acetate Ki P− inhibition constant of plasmid production by the specific growth rate Y PA/S acetate yield on glucose Y PA/G acetate yield on glycerol Y X/S biomass yield on glucose Y X/G biomass yield on glycerol Y X/A biomass yield on acetate Y P/X S plasmid yield on biomass growth on glucose Y P/X G plasmid yield on biomass growth on glycerol Y P/X A plasmid yield on biomass growth on acetate 2012, 2009a,b; Danquah and Forde, 2007; O'Kennedy et al., 2003, 2000; Wang et al., 2001), engineering the culture conditions such as temperature to increase the plasmid segregation stability (Carnes et al., 2006), plasmid engineering for example to increase its structural stability, to avoid the use of antibiotics as selective markers or to minimize its dimension (Mairhofer et al., 2010; Vidal et al., 2008; Goh and Good, 2008; Cranenburgh et al., 2001) and the host cell engineering. Concerning the E. coli genome engineering, it is worth to highlight research focusing deletions such as recA and endA to increase plasmid segregation stability (Phue et al., 2008; Zhao et al., 2007) and mutations to activate the glucose consumption while diminishing the acetate accumulation (Phue et al., 2010, 2008, 2005), or to redirect the carbon flux to the pentose phosphate pathway to enhance nucleotide synthesis and consequently plasmid production (Gonç alves et al., 2013). "
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    ABSTRACT: The need for the development of economic high plasmid production in Escherichia coli cultures is emerging, as a result of the latest advances in DNA vaccination and gene therapy. In order to contribute to achieve that, a model describing the kinetics involved in the bioproduction of plasmid by recombinant E. coli DH5α is presented, as an attempt to understand the complex and non-linear metabolic relationships and the plasmid production occurring in dynamic batch culture environments, run under different media compositions of glucose and glycerol, that result in distinct maximum biomass growths (between 8.2 and 12.8 g DCW/L) and specific plasmid productions (between 1.1 and 7.4 mg/g DCW). The model based on mass balance equations for biomass, glucose, glycerol, acetate and plasmid accurately described different culture behaviors, either using glucose or glycerol as carbon source, or mixtures of both. From the seventeen parameters obtained after model simplification, the following ten parameters were found to be independent of the carbon source composition: the substrate affinity constants, the inhibitory constants of biomass growth on glycerol by glucose, of biomass growth on acetate by glycerol and the global biomass growth by acetate, and the yields of biomass on acetate, acetate on glucose and glycerol, and plasmid on glucose. The parameters that depend on the culture composition, and that might explain the differences found between cultures, were: maximum specific growth rates on glucose, glycerol and acetate; biomass yield on glucose and glycerol; and plasmid yield on glycerol and acetate. Moreover, a crucial role of acetate in the plasmid production was revealed by the model, with most of plasmid production being associated to the acetate consumption. The model provides meaningful insight on the E. coli dynamic cell behavior concerning the plasmid bioproduction, which might lead to important guidelines for culture optimization and process scale-up and control.
    No preview · Article · Jul 2014 · Journal of Biotechnology
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