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

Abstract

BackgroundPlasmid 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.ResultsThe 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.ConclusionThe 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.

The DnaE polymerase from Deinococcus radiodurans features RecA-dependent DNA polymerase activity

“…This evidence agrees with the finding that genome replication in D. radiodurans RecA − strains is slower when compared with that observed in the wild-type counterpart [36]. Conversely, deletion of RecA does not significantly affect the growth rate of E. coli [55]. Moreover, it was demonstrated that the overexpression of recA decreases the rate of replication fork progression in E. coli [29], presumably by inhibiting αEc replisomes [28].…”

Improving Cell‐Free Protein Synthesis through Genome Engineering of Escherichia coli Lacking Release Factor 1

“…It has previously been observed that an endA deletion strain exhibits increased plasmid DNA production in vivo, [27] but its role was not clear in vitro, as the endA deletion was previously assessed only in combination with recCBD deletion. [17] In CFPS reactions performed with extracts from source strains lacking endonuclease I (MCJ.495), we observed a greater than fourfold increase in sfGFP synthesis compared to that of rEc.E13.ΔprfA (Figure 1D).…”

Modification of glucose import capacity in Escherichia coli: physiologic consequences and utility for improving DNA vaccine production

“…pHN concentration was determined by extracting plasmid DNA with the Qiagen Spin Mini Prep kit (Hilden, Germany), following manufacturer instructions. The plasmid DNA concentration was determined at 260 nm using a Nanodrop UV spectrophotometer [27]. …”