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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

ABSTRACT

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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    • "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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    • "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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    No preview · Article · Jul 2014 · Journal of Biotechnology
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