Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

Vidal, Luis; Pinsach, Jaume; Striedner, Gerald; Caminal, Glòria; Ferrer, Pau

doi:10.1016/j.jbiotec.2008.01.011

Cited by 80 publications

(57 citation statements)

References 27 publications

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“…Moreover, this expression system was further evolved into an auxotrophic marker--based plasmid maintenance system by knocking out the glyA gene from the M15 prototrophic strain and by constructing a pQE--40 derived plasmid containing the auxotrophic marker gene (glyA) under the control of a weak constitutive promoter (17). The E. coli glyA gene encodes for the enzyme serine hydroxymethyl transferase (SHMT).…”

Section: Introductionmentioning

confidence: 99%

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

et al. 2016

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Copyright and reuse:The Warwick Research Archive Portal (WRAP) makes this work by researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. A note on versions:The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher's version. Please see the 'permanent WRAP URL' above for details on accessing the published version and note that access may require a subscription. AbstractThe over--expression of proteins in recombinant host cells often requires a significant amount of resources causing an increase in the metabolic load for the host. This results in a variety of physiological responses leading to altered growth parameters, including growth inhibition. Moreover, the expression of other plasmid--encoded genes such as antibiotic resistance genes or repressor proteins may also alter growth.In this work, we have developed a second generation of an Escherichia coli expression system with an antibiotic--free plasmid maintenance mechanism based on an auxotrophic marker (glyA). Metabolic burden related to plasmid maintenance and heterologous protein expression has been minimized by tuning the expression levels of a repressor protein (LacI) and glyA using a selected library of promoters and applying synthetic biology tools that allow the rapid construction of vectors. We apply our engineered antibiotic--free expression 2 system to the FucA over--production, showing increased production levels.Our results showed that the aforementioned approaches are of paramount importance in order to increment the protein production in terms of mass and activity. Acknowledgements:This work was supported by the project "Novel Alternatives for Microbial Production of Enzymes and Multienzymatic stereoselective Synthesis (EnzProSyn)". M. P. acknowledges the Universitat Autònoma de Barcelona for the pre--doctoral grant.

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Section: Introductionmentioning

confidence: 99%

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

et al. 2016

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“…This would add to the difficulty of curing diseases caused by infectious pathogens. Accordingly, the use of antibiotic resistance genes is undesirable in many areas of biotechnology, especially in gene therapy products and genetically engineered microorganisms (17,23,28). Furthermore, the addition of antibiotics is costly in large-scale cultivation, and there are risks of contamination of the final product with antibiotics (2,3).…”

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“…The choice of the essential gene used for complementation of host auxotrophy is critical, and it is mainly involved in DNA precursor, amino acid, or cell wall biosynthetic pathways. Various essential genes, such as asd, thyA, and glnA, have been utilized to construct AC systems (5,9,21,22,24,26,28). However, all of these systems require extra nutrients or expensive reagents.…”

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Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

Dong

Xiang

Shao

2010

Appl Environ Microbiol

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The use of antibiotic resistance genes in plasmids causes potential biosafety and clinical hazards, such as the possibility of horizontal spread of resistance genes or the rapid emergence of multidrug-resistant pathogens. This paper introduces a novel auxotrophy complementation system that allowed plasmids and host cells to be effectively selected and maintained without the use of antibiotics. An Escherichia coli strain carrying a defect in NAD de novo biosynthesis was constructed by knocking out the chromosomal quinolinic acid phosphoribosyltransferase (QAPRTase) gene. The resistance gene in the plasmids was replaced by the QAPRTase gene of E. coli or the mouse. As a result, only expression of the QAPRTase gene from plasmids can complement and rescue E. coli host cells in minimal medium. This is the first time that a vertebrate gene has been used to construct a nonantibiotic selection system, and it can be widely applied in DNA vaccine and gene therapy. As

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“…Expression levels of the plasmid-borne complementation gene need to be optimized, as overexpression can lead to toxic effects (Vidal et al, 2008). Laboratory-based auxotrophies also typically rely on defined minimal media that lack the key natural metabolite; however, in deployment beyond a lab, heterogeneous environments may remove this selection pressure.…”

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confidence: 99%

Building-in biosafety for synthetic biology

2013

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As the field of synthetic biology develops, real-world applications are moving from the realms of ideas and laboratory-confined research towards implementation. A pressing concern, particularly with microbial systems, is that self-replicating re-engineered cells may produce undesired consequences if they escape or overwhelm their intended environment. To address this biosafety issue, multiple mechanisms for constraining microbial replication and horizontal gene transfer have been proposed. These include the use of host-construct dependencies such as toxin-antitoxin pairs, conditional plasmid replication or the requirement for a specific metabolite to be present for cellular function. While refactoring of the existing genetic code or tailoring of orthogonal systems, e.g. xeno nucleic acids, offers future promise of more stringent 'firewalls' between natural and synthetic cells, here we focus on what can be achieved using existing technology. The state-ofthe-art in designing for biosafety is summarized and general recommendations are made (e.g. short environmental retention times) for current synthetic biology projects to better isolate themselves against potentially negative impacts. IntroductionSynthetic biology aims to design, model and apply modular whole-cell systems to provide solutions to various challenges (Khalil & Collins, 2010). Real-world applications of synthetic biology range from molecular biosynthesis in enclosed bioreactors (Martin et al., 2003) through to sensing and acting upon external cues during environmental release, such as for biosensors (French et al., 2011), bioremediation (Singh et al., 2011) and biomining (Brune &Bayer, 2012). The majority of research and development in synthetic biology has utilized microbes as the host cells, which, in comparison with multicellular organisms, are more rapid to engineer and easier to understand. As synthetic biology advances, however, concerns are being raised about adverse effects that synthetic microbes may have if more broadly used or released into the environment (Dana et al., 2012;Moe-Behrens et al., 2013). Could genetically modified microbes (GMMs) outcompete native species and disrupt habitats? Could altered or synthetic genetic material escape its host and contaminate indigenous organisms?These concerns echo old questions raised previously by the introduction of recombinant DNA technology (Berg & Singer, 1995). At the 1975 Asilomar conference, scientists agreed on a cautious approach, incorporating both physical and biological containment into experimental design to minimize environmental risks that cisgenics or transgenics may pose (i.e. sequences native to the host, or to another species, respectively) (Berg et al., 1975). Four decades later, these principles have so far ensured no significant disaster (Berg & Singer, 1995;Benner & Sismour, 2005). Following the recent demonstration of a working synthetic genome (Gibson et al., 2010), a high-profile review has reaffirmed that the same caution applies to the use of 'syngenic' material, i.e. no...

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Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

Cited by 80 publications

References 27 publications

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

Using promoter libraries to reduce metabolic burden due to plasmid-encoded proteins in recombinant Escherichia coli

Novel Antibiotic-Free Plasmid Selection System Based on Complementation of Host Auxotrophy in the NAD De Novo Synthesis Pathway

Building-in biosafety for synthetic biology

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