Enhanced biosynthesis of plasmid DNA from Escherichia coli VH33 using Box–Behnken design associated to aromatic amino acids pathway

Martins, Luís C; Pedro, Ana; Oppolzer, David; Sousa, Fani; Queiroz, João A.; Passarinha, Luís A.

doi:10.1016/j.bej.2015.02.001

Cited by 12 publications

(10 citation statements)

References 47 publications

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“…This increase in overflow metabolism products in high temperature fermentation has been reported. Furthermore, the Y Ac/S values, obtained in the present work, were lower and can be explained by the use of glycerol instead of glucose in the fermentations [11,50]. However, the maximum acetate production observed was higher compared to the batch cultures which used glucose at 37 • C [6,51].…”

Section: Effect Of Temperature and Initial Kanamycin Concentration Oncontrasting

confidence: 57%

“…The On the other hand, cells grown at 42 • C and kanamycin 50 mg/L in micrograph Figure 3b H-T (42)(43)(44)(45)(46)(47)(48)(49)(50) showed a filamentous morphology essentially when cells elongate and replicate their DNA, but do not septate and divide. These morphological characteristics are in agreement with the ones reported by Silva et al [12].…”

Section: Cell Damage and Morphology Changes At Different Temperaturesmentioning

confidence: 98%

“…(a) A-Control: control at t = 0 for all treatments were taken at steady-state phase, B-T (30-0) , C-T (30-300) , D-T . (b) E-T (42-0) , F-T (42-300) , G-T (36-50) , H-T(42)(43)(44)(45)(46)(47)(48)(49)(50) .…”

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confidence: 99%

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

et al. 2019

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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.

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Section: Effect Of Temperature and Initial Kanamycin Concentration Oncontrasting

confidence: 57%

Section: Cell Damage and Morphology Changes At Different Temperaturesmentioning

confidence: 98%

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

et al. 2019

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“…Despite the attractive performance of the proteome-reduced strain, the Y pDNA/X are still lower than those reported using thermal induction [ 26 , 27 ]. However, there is a number of additional genetic interventions that have been reported to enhance pDNA production [ 24 , 28 , 29 ].…”

Section: Resultsmentioning

confidence: 97%

Plasmid DNA Production in Proteome-Reduced Escherichia coli

et al. 2020

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The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

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“…In E. coli and due to the higher copy number of the target gene usually achieved with plasmid-based systems, recombinant proteins are typically expressed in E. coli from medium to high plasmid copy number (PCN) based on a Col1Ederived origin of replication, (Baneyx 1999). The PCN is correlated with the recombinant gene dosage and can be accurately determined by quantitative Polymerase-Chain Reaction (qPCR) procedures (Lee et al 2006;Martins et al 2015). A recent study by Jensen et al (2017) provided a systematic approach to identify gene disruptions that increase MP expression in E. coli and can be used to improve expression of any protein that poses a cellular burden.…”

Section: Genetic-level Strategiesmentioning

confidence: 99%

Smoothing membrane protein structure determination by initial upstream stage improvements

Pedro

Queiroz

Passarinha

2019

Appl Microbiol Biotechnol

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Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

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Enhanced biosynthesis of plasmid DNA from Escherichia coli VH33 using Box–Behnken design associated to aromatic amino acids pathway

Cited by 12 publications

References 47 publications

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

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

Plasmid DNA Production in Proteome-Reduced Escherichia coli

Smoothing membrane protein structure determination by initial upstream stage improvements

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