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

Ricardo Aguilar-López,Alvaro R. Lara,Ma. del Carmen Montes-Horcasitas,Jaime Ortega-López,Fernando Grijalva-Hernández,Montserrat Escobar-Rosales,Jesús Vega-Estrada

doi:10.3390/microorganisms7120711

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.

Highlights

DNA plasmids are extrachromosomal molecules that replicate independently to the host chromosome but using their own replication machinery [1]. Plasmid DNA (pDNA) is used in vaccines and treatment of genetic diseases and cancer
Escherichia coli DH5α is the most studied commercial strain to improve the pDNA production, several strains have been developed for the same objective [3,6,7]. pDNA production is affected by host strain, plasmid type and size, genetic modification of the host strain, culture type, medium composition, and growth conditions, such as: the specific growth rate (μ); the percentage of dissolved oxygen (%DO); the increase of temperature and heating rate; and the pH control and gradients
The results allow us to provide evidence about the capacity of using high antibiotic concentration to reduce metabolic overflow products and neomycin phosphotransferase II (NPTII) synthesis related to metabolic burden

Summary

Introduction

DNA plasmids (pDNA) are extrachromosomal molecules that replicate independently to the host chromosome but using their own replication machinery [1]. pDNA is used in vaccines and treatment of genetic diseases and cancer. PDNA production is affected by host strain, plasmid type and size, genetic modification of the host strain, culture type, medium composition, and growth conditions, such as: the specific growth rate (μ); the percentage of dissolved oxygen (%DO); the increase of temperature and heating rate; and the pH control and gradients (revised by Islas-Lugo et al [8]). Organic acids production (mainly acetic acid) impact cell growth and plasmid yields during the aerobic growth of E. coli on glucose, glycerol, and other carbohydrates [9,10]. Temperature up-shift causes a heat shock response by synthesizing heat shock proteins (HSPs). These HSPs include proteases and chaperones to reduce problems of misfolded, unfolded or denatured proteins [13]

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