DNA plasmid production in different host strains of Escherichia coli

Abstract

We compared plasmid DNA production in 13 strains of Escherichia coli in shake flasks using media containing glucose or glycerol. DNA yield from either carbon source showed small correlation with maximum growth rate. Three strains, SCS1-L, BL21 and MC4100, were selected for a controlled exponential fed-batch process at a growth rate of 0.14 h(-1) to an optical density of about 70, followed by a four-hour heat treatment. Prior to heat treatment, SCS1-L generated 15.4 mg DNA/g, BL21 generated 11.0 mg DNA/g and MC4100 generated 7.9 mg DNA/g, while after heat treatment the strains attained DNA yields, respectively, of 18.0, 15.0 and 6.8 mg/g. The strains also varied in their percentage of supercoiled DNA after heat treatment, with SCS1-L averaging 66% supercoiled, BL21 17% and MC4100 40%. We further investigated the two strains that yielded the highest percentage of supercoiled DNA (SCS1-L and MC4100) at a higher growth rate of 0.28 h(-1). At this condition, a slightly lower DNA yield was generated faster, and the percentage of supercoiled DNA increased. Heat treatment improved DNA yield, and surprisingly did so to a greater extent at the higher growth rate. As a consequence of these factors, higher growth rates might be advantageous for DNA production.

Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production

“…A few high yield fed-batch processes (0.5–2.2 g/L, up to 51 mg plasmid/g DCW) have been described that enable cost effective plasmid manufacture. These processes all couple reduced growth rate during plasmid production with high copy replication origins in cells grown at 37°C (Listner et al, 2006; Huber et al, 2005b)or 42°C (Singer et al, 2009; Carnes et al, 2006; Carnes and Williams, 2006; reviewed in Carnes and Williams, 2007). Reduced growth rate increases copy number (Lin-Chao and Bremer, 1986; Seo and Bailey, 1985), % supercoiling (O’Kennedy et al, 2003) and plasmid retention.…”

Plasmid DNA fermentation strain and process‐specific effects on vector yield, quality, and transgene expression

“…A few high yield fed-batch plasmid fermentation processes (500-2,200 mg/L) have been described (Carnes et al, 2006;Listner et al, 2006;Mairhofer et al, 2010;Phue et al, 2008;Singer et al, 2009;Williams et al, 2009c). These processes all couple reduced growth rate (which generally increases copy number) with high copy replication origins (reviewed in Carnes and Williams, 2007).…”

Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode

“…The common host for pDNA production is the bacterium Escherichia coli . Several E. coli strains have been reported for pDNA production, such as DH5α [2-4], DH5 [5], DH1 [6,7], JM108 [8]; SCS1-L [9] and DH10B [10]. Most of the strains used for pDNA production are selected by its previous use in laboratory-scale protocols [11,12] and may be not suitable for process-like conditions.…”

“…Aerobic acetate production -known as overflow metabolism- results from an imbalance between glycolysis and tricarboxylic acids cycle [13,14]. Some of the strains commonly used for pDNA production present elevated overflow metabolism, including E. coli DH5α and DH1 [9]. While the conventional way of avoiding overflow metabolism is reducing the glucose uptake in the so called fed-batch mode, the constant supply of glucose to the bioreactor requires additional equipment, results in a decrease of growth rate and frequently causes substrate gradients at the feeding zone in production bioreactors that trigger undesirable physiological effects [15-17].…”