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Duarte Miguel F. Prazeres is full professor at the iBBâInstitute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior TÃ©cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Claudia P.A. Alves is a research assistant at iBBâInstitute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior TÃ©cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Sofia O. D. Duarte is a PhD student at the iBBâInstitute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior TÃ©cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
Gabriel A. Monteiro is associate professor at the iBBâInstitute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior TÃ©cnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
The authors describe the impact of the knocking of the pgi gene of the wild type MG1655 strain on the growth kinetics of plasmid-free and plasmid-bearing cells.
The possibility of using plasmids as biopharmaceuticals for gene therapy and DNA vaccination has gradually emerged during the past 20 years (1). The plasmid DNA (pDNA) molecules in these biopharmaceuticals should transfer genes to target individuals (humans and animals) to prevent or exercise control over diseases such as AIDS, tuberculosis, and cancer. A significant challenge in this context is the development of manufacturing processes capable of producing the required material to run pre-clinical and clinical trials (1, 2).
The manufacturing of pDNA comprises a series of interlinked activities (see Figure 1) designed to consistently obtain a defined amount of a safe and effective product (1). The pDNA is typically produced by replication in Gram-negative Escherichia coli. However, in most cases, the strains used (e.g., DH5 α, JM101, BL21) were originally developed for cloning or for the production of recombinant proteins (1). Due to their miscellaneous mutagenized genetic backgrounds, such strains may thus not be the best choice for producing the large amounts of pDNA required for clinical trials and eventual commercialization (3, 4). A more rational approach is to start from a wild-type strain and select/mutate genes that are likely to have an impact on the kinetics of cell growth and on the synthesis of pDNA (5, 6). Such engineered strains should grow up to high cell densities and produce large quantities of pDNA (i.e., maximize volumetric pDNA yield, mg pDNA/L) as quickly as possible and at the lowest cost. Although the growth/production medium, bioreactor operating variables, and culture strategies are key aspects at this stage, starting from a robust, high producer of pDNA is highly recommended.
Figure 1: Overview of main activities in plasmid manufacturing. [Courtesy of authors]
This article describes the growth kinetics, pH profile, and pDNA volumetric yield obtained with GALG20, an endA, recA, and pgi knockout of the wild type MG1655 strain (6, 7). One of the targets, pgi, codes for phosphoglucose isomerase, an enzyme that catalyzes the conversion of glucose-6-phosphate into fructose-6-phosphate. This knockout allows the redirection of the carbon flux to the pentose phosphate pathway, leading to an increase in nucleotide synthesis and pDNA production, whereas the deletion of endA and recA minimizes pDNA nonspecific digestion and recombination (6). Additionally, the authors show that supercoiled (sc) pDNA produced by these cells can be isolated from impurities and from open circular (oc) pDNA by an optimized hydrophobic interaction chromatography (HIC) step.
MATERIALS AND METHODSStrains and plasmids
The GALG20 strain was constructed by deleting the genes endA, recA, and pgi in the wild type strain MG1655 by P1 transduction as described previously (6). The strain was then transformed with the 3697 bp plasmid pVAX1GFP (8).
Inocula were prepared from frozen stocks of transformed GALG20 and wild type MG1655 strains in 15-mL conical centrifuge tubes with 5 mL of LB medium (NZYTech, Lisbon, Portugal) supplemented, when required, with 30 µg/mL kanamycin (Amresco, Solon, OH). Cells were incubated overnight at 37 ºC and 250 rpm and used to inoculate 250-mL baffled shake flasks containing 50 mL of complex medium (20 g/L glucose, 10 g/L bacto peptone, 10 g/L yeast extract, 3 g/L ammonium sulfate ((NH4)2SO4), 3.5 g/L potassium hydrogen phosphate (K2HPO4), 3.5 g/L potassium dihydrogen phosphate (KH2PO4), 200 mg/L thiamine, 2 g/L magnesium sulfate (MgSO4), and 1 mL/L of a trace element solution ) with 30 µg/mL kanamycin, pH 7.0, at an initial optical density at 600 nm (OD600) of approximately 0.1.
Transformed GALG20 cells were harvested after 10 hours by centrifugation and subjected to alkaline lysis as described previously (7). Then, plasmid in the clarified lysates was precipitated with 0.7 volumes of pure isopropanol (2 hours, -20 °C) and recovered by centrifugation (30 min at 18,514 g and 4 °C). After drying at 4 ºC, pellets were resuspended in 10 mM Tris-HCl, pH 8, and solid ammonium sulfate was added up to a concentration of 2.5 M to precipitate proteins (15 min on ice) and condition the solution in preparation for HIC. Precipitated proteins were removed by centrifugation (30 min, 17,949 g, 4 °C). The pDNA in this solution was purified by HIC using a column packed with 10 mL of Phenyl Sepharose 6 Fast Flow resin in an ÄKTApurifier100 system (GE Healthcare). A mobile phase containing mixtures of 2.2 M ammonium sulfate in 10 mM Tris-HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8 (buffer A) and 10 mM Tris-HCl, 1 mM EDTA, pH 8 (buffer B) was used to run the separation. The absorbance of the eluate was continuously measured at 254 nm with a UV detector positioned at the column outlet, and the system was operated at 2 mL/min. Following column equilibration with three column volumes (CV) of 17% buffer B (≈ 204 mS/cm), 1 mL of the pDNA-containing feed was injected. Unbound material was washed out with 4 CV of 17% B, and two elution steps were performed with 2 CV of 35% B (≈ 173 mS/cm) and with 2 CV of 100% B (≈ 2 mS/cm). Fractions (1.5 mL) corresponding to peaks were collected during the run and dialyzed against 10 mM Tris-HCl, 1 mM EDTA, pH 8 for desalting prior to analysis in 1% (w/v) agarose gels.
Analytical chromatography was performed in an ÄKTApurifier10 system (GE Healthcare), using a commercial Tricorn high-performance column with a 1.7 mL bed volume (SOURCE 15PHE 4.6/100 PE, GE Healthcare) and following a modification of the HIC-HPLC (high-performance liquid chromatography) method described by Diogo et al. (10). Briefly, after column equilibration (2.5 min) with 1.5 M ammonium sulfate in 10 mM Tris-HCl pH 8, 50 µL samples were injected and elution was performed for 1 min with the same buffer. Species bound to the matrix were then eluted with 10 mM Tris-HCl pH 8 for 0.8 min and column was re-equilibrated for 5.5 min with the initial buffer. The absorbance of the eluate was continuously measured at 260 nm, and the system was operated at 1 mL/min. The plasmid was quantified using a calibration curve constructed with plasmids standards (purified using the HiSpeed plasmid Maxi Kit from Qiagen) prepared in a concentration range from 0 to 100 µg/mL.
Agarose gels were prepared with 1% (w/v) agarose (ThermoFisher Scientific) in tris-acetate-EDTA (TAE) buffer (40 mM Trisbase, 20 mM acetic acid and 1 mM EDTA, pH 8) and loaded with samples mixed with a 6X loading buffer (40% w/v sucrose, 0.25% w/v bromophenol blue), using NZYDNA ladder III (NZYTech, Lisbon, Portugal) as molecular weight marker. Electrophoresis was run at 120 V for 90 min, using 1% TAE as the running buffer. Gels were stained in an ethidium bromide solution (0.4 µg/mL), and images were obtained with an Eagle Eye II gel documentation system (Stratagene, La Jolla, CA).
The pgi gene of the wild type E. coli strain MG1655 was knocked out with the goal of redirecting the carbon flux into the pentose phosphate pathway to increase nucleotide synthesis, NADPH generation, and hopefully pDNA production (5). The growth characteristics of the new strain GALG20 (either non-transformed or transformed) were studied and compared with MG1655 (see Figure 2). Experiments were performed in baffled shake flasks using 20 g/L glucose in a rich medium at an initial pH of 7.0. The pH of the medium was also measured during the course of cell growth (see Figure 3). The data show that during the first four hours, there are no differences between growth profiles of the native MG1655 and GALG20 strains. A significant drop of the pH of the medium to 5.7, however, is detected at four hours for the case of MG1655 that is in stark contrast with the lack of pH variation observed for GALG20 (pH ~ 6.9). From the fourth hour on, GALG20 continued to grow at a rate that was significantly higher when compared with MG1655. As a result, optical densities (ODs) of 30 were obtained for non-transformed GALG20 after 10 h, whereas MG1655 did not surpass ODs of 10 at the same time instant. After 10 h of growth, pH values decreased to 4.8 for MG1655, 5.6 for non-transformed GALG20, and 6.3 for transformed GALG20. The plasmid DNA produced by transformed GALG20 cells was quantified by HIC-HPLC from clarified alkaline lysates of cells harvested after 10 h of growth as described by Gonçalves et al. (7). Results from four independent shake flask cultures indicate a pDNA production of 63.0 ± 11.7 mg/L.
Figure 2: Growth curves of Escherichia coli strains GALG20 and MG1655. Complex medium with 20 g/L glucose at pH 7 was used to grow cells in baffled shake flasks (37 ËC, 250 rpm). Results are an average of three independent experiments. [Courtesy of authors]
Figure 3: Variation of medium pH during growth of Escherichia coli strains GALG20 and MG1655. Complex medium with 20 g/L glucose at pH 7 was used to grow cells in baffled shake flasks (37 ËC, 250 rpm). Results are an average of three independent experiments. [Courtesy of authors]
Supercoiled pDNA isolation
Experiments were performed to check if the knockout of the pgi gene had an impact on the purification and final quality of pDNA. Firstly, transformed GALG20 cells grown as described above were disrupted by alkaline lysis to release pDNA. Then, sequential precipitation with isopropanol and with ammonium sulfate was used to concentrate nucleic acids and remove protein and RNA impurities, respectively (11). The resulting high-salt solution (~2.5 M) was then subjected to HIC (see Figure 4) using a phenyl Sepharose column to isolate the sc isoform from the mixture containing sc and oc pDNA and also RNA (see lane F in Figure 5). Elution steps with decreasing ammonium sulfate concentrations were used to separate plasmid topoisomers and RNA. The chromatogram (Figure 4) is characterized by two flowthrough peaks emerging sequentially at 17% B (~1.83 M ammonium sulfate), a first elution peak at 35% B (~1.43 M ammonium sulfate), and a second peak at 100% B (0 M ammonium sulfate). An agarose gel electrophoresis analysis of the corresponding fractions shows clearly that the flowthrough contains oc pDNA (lanes 3 and 8, Figure 5), whereas sc DNA is obtained in the elution peak at 35% B (lane 37, Figure 5). As for RNA, it is removed only when the column is eluted at a low salt concentration (lane 53, Figure 5).
The pgi knockout strain GALG20 is able to grow up to optical densities that are significantly higher when compared with the MG1655 control strain (~30 vs. ~10 after 10 h). Furthermore, the acidification of the culture medium during growth of GALG20 is less pronounced when compared with MG1655. The sharp decrease of pH in the case of MG1655 is consistent with acetate production, a phenomenon that occurs in aerobic conditions when high concentrations of glucose inhibit respiration (Crabtree effect). In the case of GALG20, however, the results indicate that the knocking out of pgi impaired the ability of the new strain to produce acetic acid (and hence acidify the medium). This result is consistent with the down-regulation of glycolysis and of the tricarboxylic acid cycle, and with the redirection of the carbon flux to the pentose phosphate pathway. Although the main purpose of the aforementioned knockout was the increase in nucleotide synthesis and, consequently, in pDNA production, the low acetate production by the GALG20 strain makes it possible for cells to reach a higher density (≈ 3-fold higher than the wild type MG1655), especially when medium pH is not controlled during cell growth. The results obtained for pDNA volumetric yield (63.0 ± 11.7 mg/L) also prove the effect of the pgi knockout on pDNA production. At shake-flask scale, the volumetric yield of GALG20 is 7- to 10-fold higher (7) than the one presented by its parental strain MG1655 also deleted for the endA and recA genes (MG1655ΔendAΔrecA), highlighting the favorable outcome of the metabolic pathway redirection imposed by the pgi knockout.
Supercoiled plasmid DNA produced by GALG20 was isolated and purified by a process that combines alkaline lysis with tandem precipitation with isopropanol, ammonium sulfate and purification by HIC. Agarose gel analysis of collected fractions show that the method applied is able to separate sc pDNA from the oc isoform and from RNA. Densitometry analysis of the bands in the agarose gel presented in Figure 5 confirmed the successful isolation of sc pDNA. The column feed contained approximately 51.3% of sc pDNA and 48.7% of oc pDNA (lane F). The oc pDNA was removed essentially in the first (lane 3) and second (lane 8) flowthrough peaks. A small amount of sc pDNA was lost in the second flowthrough peak (2.4% of the total pDNA present, see lane 8). The elution of the sc pDNA isoform occurred mainly during the step at 35% B (~1.43 M ammonium sulfate). An analysis of the corresponding fraction (lane 37) shows that 99.2% of the pDNA recovered is sc, a level of homogeneity that is superior to the FDA requirements for clinical-grade pDNA vectors (12).
This work highlights the advantages of engineering E.coli strains for improved pDNA production as a mean to develop efficient manufacturing processes able to meet pre-clinical and clinical trial requirements. Specifically, the authors present a pgi knockout E.coli strain (GALG20) that is able to reach higher cell densities and pDNA yields than its parental strain MG1655, as a consequence of a metabolic pathway redirection. Additionally, the decrease in the pH of GALG20 cell cultures was less pronounced when compared with the variation observed for the wild type MG1655. These results are especially interesting when carrying cultures with no external pH control, since impairment of cell growth is reduced. In addition, the authors present a purification method relying on HIC that is able to isolate the therapeutically valuable sc pDNA isoform, which is virtually free from RNA and oc pDNA.
Funding received by iBB-Institute for Bioengineering and Biosciences from FCT-Portuguese Foundation for Science and Technology (UID/BIO/04565/2013 and doctoral grant SFRH/BD/84267/2012 awarded to Sofia Duarte), from Programa Operacional Regional de Lisboa 2020 (Project N. 007317), and from the European Project INTENSO (FP7-KBBE-2012-6) is acknowledged.
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Article DetailsBioPharm International
Vol. 29, No. 2
When referring to this article, please cite it as C. Alves et al., "Use of an E. coli pgi Knockout Strain as a Plasmid Producer," BioPharm International 29 (2) 2016.