Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Bentley, William E.; Mirjalili, Noushin; Andersen, Dana C.; Davis, Robert H.; Kompala, Dhinakar S.; Bentley, Introduction by William E.

doi:10.1002/bit.22292

Cited by 74 publications

(71 citation statements)

References 9 publications

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“…At the beginning of the process, the cell growth and consumption of glycerol showed a little slower than those of the wild type strain, implying a metabolic burden placed on by the plasmid [20]. However, from Fig.…”

Section: Resultsmentioning

confidence: 83%

“…Just as KG1 (pUC18 K-dhaT), the growth and consumption of glycerol were slower than those of the wild type strain (Fig. 5), because of the metabolic burden placed on by the plasmid [20]. But during the process of fermentation no apparent pause was observed.…”

Section: Resultsmentioning

confidence: 99%

“…At first, we assumed the growth stagnation was due to the over-expression of plasmidencoded protein, since a large amount of energy was consumed during protein synthesis [20]. But it was difficult to explain the recovery without loss of plasmids or inserts.…”

Section: Resultsmentioning

confidence: 99%

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Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by Klebsiella pneumoniae under micro-aerobic conditions

Zhao

Zheng

et al. 2008

Bioprocess Biosyst Eng

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Glycerol dehydrogenase (GDH) and 1,3-propanediol (1,3-PD) oxidoreductase had been proved two key enzymes for 1,3-PD production by Klebsiella pneumoniae. Fed-batch fermentations of the recombinant K. pneumoniae strains, over-expressing the two enzymes individually, were carried out under micro-aerobic conditions, and the behaviors of the recombinants were investigated. Results showed that over-expression of 1,3-PD oxidoreductase did not affect the concentration of 1,3-PD. However, it enhanced the molar yield from 50.6 to 64.0% and reduced the concentration of by-products. Among them, the concentrations of lactic acid, ethanol and succinic acid were decreased by 51.8, 50.6 and 47.4%, respectively. Moreover, in the recombinant the maximal concentration of 3-hydroxypropionaldehyde decreased by 73.6%. Over-expression of GDH decreased the yield of ethanol and 2,3-butanediol, meanwhile it increased the concentration of acetic acid. No significant changes were observed both in 1,3-PD yield and glycerol flux distributed to oxidative branch.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by Klebsiella pneumoniae under micro-aerobic conditions

Zhao

Zheng

et al. 2008

Bioprocess Biosyst Eng

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“…Escherichia coli is the most preferred host for expressing recombinant proteins due to several advantages like media, faster growth and easy fermentation [12]. E. coli BL21 (DE3) is widely used for recombinant protein over-production [7,8,27], however, the limitation includes formation of inclusion body, high cost and toxicity of IPTG [21]. To overcome these limitations, osmotically inducible E. coli GJ1158 host with salt (NaCl) as inducer was developed for over production of recombinant proteins cloned in T7 vectors [9].…”

Section: Introductionmentioning

confidence: 99%

Production, purification and diagnostic application of filarial recombinant protein WbSXP-1 expressed in salt inducible Escherichia coli

Janardhan

Pandiaraja

Thirugnanam

et al. 2007

J Ind Microbiol Biotechnol

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Wuchereria bancrofti protein WbSXP-1 was identified and established as a potential candidate for the diagnosis of lymphatic filariasis. For the economic production of rWbSXP-1, osmotically (salt) inducible Escherichia coli GJ1158 was preferred. Cultivation and expression was optimized in 3 L airlift bioreactor (ALB) and was successfully extended to 30 L ALB. Purification of rWbSXP-1 his-tag protein was optimized in technical scale using FPLC and the maximal recovery of rWbSXP-1 with significant level of purity was achieved using the combination of IMAC and gel filtration. Quality criteria for immuno-reactivity of purified rWbSXP-1 were established for diagnostic applications. Enhancement of sensitivity in rapid diagnostic format was optimized to effectively detect weak to strong antibody reactivity in individuals exposed to lymphatic filariasis. Performance of the rapid format during field evaluation was successful. The accelerated stability assessment of the rapid format satisfied the requirements of WHO-cGMP norms. This investigation presents a successful technical scale production and purification of rWbSXP-1 considering the future industrial application and an enhanced rapid flow through antibody assay for the diagnosis of human lymphatic filariasis.

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“…In times using strong host/vector systems to gain maximal expression of recombinant proteins the cells are quickly overburdened [3] due to the imbalance of formation of foreign and cellular protein and/or interaction of the foreign protein with the host cell metabolism [4]. As a consequence, the global stress response molecule guanosine tetraphosphate (ppGpp) is formed which in turn triggers global changes of protein expression program, e.g.…”

Section: Introductionmentioning

confidence: 99%

Design of transcriptional fusions of stress sensitive promoters and GFP to monitor the overburden of Escherichia coli hosts during recombinant protein production

Nemecek

Marisch

Juric

et al. 2007

Bioprocess Biosyst Eng

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Due to the lack of appropriate sensors for monitoring changes of Escherichia coli cells and the huge complexity of cellular systems, many of the present protein production processes are still far from optimal. Aiming at maximal exploitation of the host cell, enhanced knowledge of cellular reactions related to recombinant protein expression is required. Current methods like DNA microarrays and 2-D-electrophoresis enable the acquisition of transcriptional and translational activity shifts in stress situations like heat shock, general stress response, nutrient limitation, and stress caused by overexpression of heterologous proteins. However, these techniques and data processing are time consuming, therefore, the goal is to create new on-line systems such as stress promoter GFP fusions to monitor metabolic alterations. The fluorescence signal of expressed GFP can be measured by 2-D-multi-wavelength fluorescence spectroscopy, thereby allowing non-invasive on-line in vivo monitoring. Results of efficient stress monitoring approaches in ongoing protein production process are presented.

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Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria

Cited by 74 publications

References 9 publications

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by Klebsiella pneumoniae under micro-aerobic conditions

Effects of over-expression of glycerol dehydrogenase and 1,3-propanediol oxidoreductase on bioconversion of glycerol into 1,3-propandediol by Klebsiella pneumoniae under micro-aerobic conditions

Production, purification and diagnostic application of filarial recombinant protein WbSXP-1 expressed in salt inducible Escherichia coli

Design of transcriptional fusions of stress sensitive promoters and GFP to monitor the overburden of Escherichia coli hosts during recombinant protein production

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