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

Gheorghe M Borja,Blanca Barrón,Alvaro R Lara,Guillermo Gosset,Eugenio Meza Mora,Octavio T Ramírez

doi:10.1186/1475-2859-11-132

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.

Highlights

Plasmid DNA is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode
Several E. coli strains have been reported for pDNA production, such as DH5α [2,3,4], DH5 [5], DH1 [6,7], JM108 [8]; SCS1-L [9] and DH10B [10]
The typical challenges for high cell-density cultivations (HCDC) of E. coli remain as obstacles for the fast and efficient production of pDNA

Summary

Introduction

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. 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. 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 [15,16,17]. We have previously demonstrated that the substitution of the natural glucose transport system (PTS) by a constitutively expressed galactose permease under the strong trc promoter in E. coli allows efficient growth by reducing the glucose uptake rate and decreasing acetate production [18,19]. The possibility of cultivating VH33 strains and derivatives at high celldensity in batch mode is a simple and valuable alternative to fed-batch mode for the fast and efficient production of pDNA both, at early-stages of product development and at technical scale

Methods

Results

Conclusion