Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants

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doi:10.2903/j.efsa.2004.48

Cited by 19 publications

(6 citation statements)

References 63 publications

(43 reference statements)

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“…Sandoval-Vargas et al (2019) have also recently reported the use of ptxD as a selectable marker for the C. reinhardtii chloroplast, and our combined work adds a useful dominant marker to the molecular toolbox (Esland et al 2018). Importantly, this marker is not derived from a bacterial antibiotic resistance gene and therefore raises less concerns regarding GM regulation (EFSA GMO Panel 2004;Beacham et al 2017). Finally, selection of transformants based on phosphite metabolism does not suffer from false-positive issues as spontaneous mutants able to oxidise phosphite are very unlikely to arise.…”

Section: Discussionmentioning

confidence: 87%

“…Such a metabolic marker is superior to the frequently used antibiotic resistance markers since selection uses a cheap substrate (Phi) and eliminates the problem of 'false positives' since the spontaneous acquisition of Phi metabolism is not possible. Furthermore, the use of metabolic markers helps address the regulatory and safety concerns associated with the environmental spread of antibiotic resistance genes by horizontal gene transfer (HGT) during commercial cultivation (EFSA GMO Panel 2004;Beacham et al 2017). Various reports have shown that ptxD can serve as an efficient marker for generation of transgenic plants (López-Arredondo and Herrera-Estrella 2013; Nahampun et al 2016;Pandeya et al 2017), yeasts (Kanda et al 2014) and cyanobacteria (Selão et al 2019).…”

Section: Introductionmentioning

confidence: 99%

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The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Changko

Rajakumar

Young

et al. 2019

Appl Microbiol Biotechnol

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Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during setup and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bioavailable form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

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Section: Discussionmentioning

confidence: 87%

Section: Introductionmentioning

confidence: 99%

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Changko

Rajakumar

Young

et al. 2019

Appl Microbiol Biotechnol

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“…The C. reinhardtii chloroplast is actively being explored as a GRAS (‘Generally Recognised As Safe’) and sustainable platform for the commercial production of recombinant proteins [ 4 , 5 , 37 ]. Nevertheless, the presence within the engineered chloroplast of multiple copies of an antibiotic resistance marker is undesirable, both from a regulatory [ 7 ] and a metabolic burden perspective [ 8 ]. ‘Marker-free’ strategies based on the phenotypic rescue of a mutation within the plastome are preferable, and various photosynthetic mutants with point mutations or deletions in chloroplast genes have been used for the selection of transformants [ 8 ].…”

Section: Discussionmentioning

confidence: 99%

“…One key consideration of any microbial cell platform used in biotechnology is the presence in the genome of transgenes encoding antibiotic resistance that are introduced as selectable markers during transgenesis, and could spread to other microorganisms via horizontal gene transfer [ 7 ]. Given the chloroplast’s evolutionary history as an endosymbiont of an early cyanobacterial cell, such antibiotic-based selectable markers designed for high-level expression in the chloroplast are often functional in bacteria [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii

Taunt,

Jackson,

Gunnarsson

et al. 2023

Microorganisms

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‘Marker-free’ strategies for creating transgenic microorganisms avoid the issue of potential transmission of antibiotic resistance genes to other microorganisms. An already-established strategy for engineering the chloroplast genome (=plastome) of the green microalga Chlamydomonas reinhardtii involves the restoration of photosynthetic function using a recipient strain carrying a plastome mutation in a key photosynthesis gene. Selection for transformant colonies is carried out on minimal media, such that only those cells in which the mutated gene has been replaced with a wild-type copy carried on the transgenic DNA are capable of phototrophic growth. However, this approach can suffer from issues of efficiency due to the slow growth of C. reinhardtii on minimal media and the slow die-back of the untransformed lawn of cells when using mutant strains with a limited photosensitivity phenotype. Furthermore, such phototrophic rescue has tended to rely on existing mutants that are not necessarily ideal for transformation and targeted transgene insertion: Mutants carrying point mutations can easily revert, and those with deletions that do not extend to the intended transgene insertion site can give rise to a sub-population of rescued lines that lack the transgene. In order to improve and accelerate the transformation pipeline for C. reinhardtii, we have created a novel recipient line, HNT6, carrying an engineered deletion in exon 3 of psaA, which encodes one of the core subunits of photosystem I (PSI). Such PSI mutants are highly light-sensitive allowing faster recovery of transformant colonies by selecting for light-tolerance on acetate-containing media, rather than phototrophic growth on minimal media. The deletion extends to a site upstream of psaA-3 that serves as a neutral locus for transgene insertion, thereby ensuring that all of the recovered colonies are transformants containing the transgene. We demonstrate the application of HNT6 using a luciferase reporter.

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“…For instance, the large-scale cultivation of GM-plants, i.e., on about 170 million hectares worldwide (James, 2012), results in multitudinous opportunities for bacterial exposure to recombinant DNA and therefore, opportunities for unintended horizontal dissemination of transgenes (EFSA, 2004, 2009; Nielsen et al, 2005; Levy-Booth et al, 2007; Wögerbauer, 2007; Pietramellara et al, 2009; Brigulla and Wackernagel, 2010). In laboratory settings, experimental studies have demonstrated that single bacterial species can take up and recombine with DNA fragments from GM-plants under optimized conditions (e.g., Gebhard and Smalla, 1998; de Vries et al, 2001; Kay et al, 2002; Ceccherini et al, 2003).…”

Section: Introduction To Hgt In Bacterial Populationsmentioning

confidence: 99%

Detecting rare gene transfer events in bacterial populations

2014

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Horizontal gene transfer (HGT) enables bacteria to access, share, and recombine genetic variation, resulting in genetic diversity that cannot be obtained through mutational processes alone. In most cases, the observation of evolutionary successful HGT events relies on the outcome of initially rare events that lead to novel functions in the new host, and that exhibit a positive effect on host fitness. Conversely, the large majority of HGT events occurring in bacterial populations will go undetected due to lack of replication success of transformants. Moreover, other HGT events that would be highly beneficial to new hosts can fail to ensue due to lack of physical proximity to the donor organism, lack of a suitable gene transfer mechanism, genetic compatibility, and stochasticity in tempo-spatial occurrence. Experimental attempts to detect HGT events in bacterial populations have typically focused on the transformed cells or their immediate offspring. However, rare HGT events occurring in large and structured populations are unlikely to reach relative population sizes that will allow their immediate identification; the exception being the unusually strong positive selection conferred by antibiotics. Most HGT events are not expected to alter the likelihood of host survival to such an extreme extent, and will confer only minor changes in host fitness. Due to the large population sizes of bacteria and the time scales involved, the process and outcome of HGT are often not amenable to experimental investigation. Population genetic modeling of the growth dynamics of bacteria with differing HGT rates and resulting fitness changes is therefore necessary to guide sampling design and predict realistic time frames for detection of HGT, as it occurs in laboratory or natural settings. Here we review the key population genetic parameters, consider their complexity and highlight knowledge gaps for further research.

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Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants

Cited by 19 publications

References 63 publications

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Accelerating Chloroplast Engineering: A New System for Rapid Generation of Marker-Free Transplastomic Lines of Chlamydomonas reinhardtii

Detecting rare gene transfer events in bacterial populations

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