High Kanamycin Concentration as Another Stress Factor Additional to Temperature to Increase pDNA Production in E. coli DH5α Batch and Fed-Batch Cultures

Abstract

Plasmid DNA (pDNA) vaccines require high supercoiled-pDNA doses (milligrams) to achieve an adequate immune response. Therefore, processes development to obtain high pDNA yields and productivity is crucial. pDNA production is affected by several factors including culture type, medium composition, and growth conditions. We evaluated the effect of kanamycin concentration and temperature on pDNA production, overflow metabolism (organic acids) and metabolic burden (neomycin phosphotransferase II) in batch and fed-batch cultures of Escherichia coli DH5α-pVAX1-NH36. Results indicated that high kanamycin concentration increases the volumetric productivity, volumetric and specific yields of pDNA when batch cultures were carried out at 42 °C, and overflow metabolism reduced but metabolic burden increased. Micrographs taken with a scanning electron microscope (SEM) were analyzed, showing important morphological changes. The high kanamycin concentration (300 mg/L) was evaluated in high cell density culture (50 gDCW/L), which was reached using a fed-batch culture with temperature increase by controlling heating and growth rates. The pDNA volumetric yield and productivity were 759 mg/L and 31.19 mg/L/h, respectively, two-fold greater than the control with a kanamycin concentration of 50 mg/L. A stress-based process simultaneously caused by temperature and high kanamycin concentration can be successfully applied to increase pDNA production.

Sulfate limitation increases specific plasmid DNA yield and productivity in E. coli fed-batch processes

“…fed-batch [ 63 ] 31.2 * 1923 72.4 ‡ NA 62 ‡* NA pUC Ampicillin BL21 recA Lin. fed-batch [ 64 ] 15.5 759 31.2 426 § 49 ‡ 3.9 pUC Kanamycin DH5 Exp. fed-batch [ 8 ] 25.9 939 35.1 1142 36 ‡ 3.5 ColE1 None JM108 murselect Exp.…”

Antibacterial activity and mechanism of a novel bacteriocin produced by Lactiplantibacillus plantarum against Escherichia coli and Staphylococcus aureus

“…The morphology of E. coli and S. aureus was observed by SEM according to the previous method with a slight modification (Grijalva‐Hernández et al ., 2019). In the experimental groups, the E. coli and S. aureus suspension (10 mL, OD 600 = 0.6) were mixed with an equal volume of the L. plantarum CFS (10 mL) and cultured at 37°C for 12 h. The bacteria cell suspension was centrifuged with 8000 g at 4 °C for 15 min, and then the collected cells were washed three times with sterile saline (0.85% w/v).…”