High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification

Barnard, Gavin C.; Henderson, Grant E.; Srinivasan, Sriram; Gerngross, Tillman U.

doi:10.1016/j.pep.2004.09.001

Cited by 59 publications

(36 citation statements)

References 22 publications

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“…Other hosts that have been exploited for RPP include Pseudomonas putida for antibody fragments [34] and Ralstonia eutropha for organophosphohydrolase production [9]. Many more bacterial systems are described, but more than often the limited information about their genetics or metabolism or the unavailability of expression vectors or promoter systems hinders the expansion of their application.…”

Section: Bacteriamentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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Different hosts have been used for recombinant protein production, ranging from simple bacteria, such as Escherichia coli and Bacillus subtilis, to more advanced eukaryotes as Saccharomyces cerevisiae and Pichia pastoris, to very complex insect and animal cells. All have their advantages and drawbacks and not one seems to be the perfect host for all purposes. In this review we compare the characteristics of all hosts used in commercial applications of recombinant protein production, both in the area of biopharmaceuticals and industrial enzymes. Although the bacterium E. coli remains a very often used organism, several drawbacks limit its possibility to be the first-choice host. Furthermore, we show what E. coli strains are typically used in high cell density cultivations and compare their genetic and physiological differences. In addition, we summarize the research efforts that have been done to improve yields of heterologous protein in E. coli, to reduce acetate formation, to secrete the recombinant protein into the periplasm or extracellular milieu, and to perform post-translational modifications. We conclude that great progress has been made in the incorporation of eukaryotic features into E. coli, which might allow the bacterium to regain its first-choice status, on the condition that these research efforts continue to gain momentum.

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Section: Bacteriamentioning

confidence: 99%

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Waegeman¹,

Soetaert²

2011

J Ind Microbiol Biotechnol

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“…In general, low-cost recombinant proteins can be produced in high cell density fermentations (Barnard et al, 2004;Hartley, 2006;Zhang et al, 2006). Low-cost enzyme purification can be conducted by scalable purification means, such as precipitation (Banki et al, 2005;Scopes, 1993), heat precipitation (Blumer-Schuette et al, 2008;Wang and Zhang, 2009b), or adsorption/desorption (Hong et al, 2008a,b).…”

Section: Synthetic Pathway Biotransformation (Sypab)mentioning

confidence: 99%

“…Clair et al, 2000;Vasic-Racki, 2006). Second, the Y E E=X value can be increased from 0.1 to 0.3 or higher through (i) the production of over-expressed recombinant enzymes (Barnard et al, 2004;Hartley, 2006;Sørensen and Mortensen, 2005;Wang and Zhang, 2009b (ii) the use of high-cell density fermentation plus low-cost fermentation media (Barnard et al, 2004;Shiloach and Fass, 2005), and (iii) the production of high-level secretory enzymes (Choi and Lee, 2004;Hong et al, 2008b;Zhang et al, 2006). Third, the combined cost coefficient of enzyme purification and immobilization ( F P F S ) can be decreased from 20 to 5 or lower by (i) simple adsorption/desorption (Barnard et al, 2005;Hong et al, 2008a,b) or simple heat precipitation (Wang and Zhang, 2009b), (ii) direct immobilization of dead microbes that contain active enzyme (Bhosale et al, 1996), and (iii) low-cost immobilization technologies (Mateo et al, 2001;Pessela et al, 2003).…”

Section: Cost Analysis and Perspectivesmentioning

confidence: 99%

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Zhang

2010

Biotech & Bioengineering

164

137

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Cell-free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell-free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low-value products where costs of feedstock carbohydrates often account for $30-70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar-to-biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low-value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low-cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight-based total turn-over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent.

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“…The phage RNA polymerase can also generate very long mRNAs and is poorly terminated by unrelated transcription terminators [31]. Since the original publication of Studier and Moffatt, the T7 expression system has been adapted to mammalian cells and several bacteria [3,6,10,12,16,21,22]. Lussier et al [29] have developed a bifunctional vector, pFX583, that allows T7 RNApol-directed transcription in E. coli and in the filamentous Gram-positive bacterium Streptomyces lividans.…”

Section: Introductionmentioning

confidence: 99%

Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector

Lussier

Chambenoit

Côté

et al. 2010

J Ind Microbiol Biotechnol

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The metagenomic approach has greatly accelerated the discovery of new enzymes by giving access to the genetic potential of microorganisms from various environments. Function-based screening depends on adequate expression of the foreign genes in the heterologous host, which can be challenging in large-insert libraries. In this study, the shuttle cosmid vector pFX583 was used for the construction and screening of a metagenomic library. This vector allows T7 RNA polymerase-directed transcription of the cloned DNA and can be used in Escherichia coli and Streptomyces lividans. The DNA used for the library construction was obtained from an enriched biomass. The library was screened for lipolytic and proteolytic activities using E. coli and S. lividans as hosts. Numerous E. coli clones with lipolytic activity were detected. Unfortunately, proteases could not be detected in both hosts. From the lipolytic activity screen, a gene coding for a new lipase was isolated, and partial characterization was conducted.

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High level recombinant protein expression in Ralstonia eutropha using T7 RNA polymerase based amplification

Cited by 59 publications

References 22 publications

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Increasing recombinant protein production in Escherichia coli through metabolic and genetic engineering

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Construction and functional screening of a metagenomic library using a T7 RNA polymerase-based expression cosmid vector

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