Risk mitigation of genetically modified bacteria and plants designed for bioremediation

While the possible advantages of bioremediation and phytoremediation, by both recombinant microbes and plants, have been extensively reviewed, the biosafety concerns have been less extensively treated. This article reviews the possible risks associated with the use of recombinant bacteria and plants for bioremediation, with particular emphasis on ways in which molecular genetics could contribute to risk mitigation. For example, genetic techniques exist that permit the site-specific excision of unnecessary DNA, so that only the transgenes of interest remain. Other mechanisms exist whereby the recombinant plants or bacteria contain conditional suicide genes that may be activated under certain conditions. These methods act to prevent the spread and survival of the transgenic bacteria or plants in the environment, and to prevent horizontal gene flow to wild or cultivated relatives. Ways in which these genetic technologies may be applied to risk mitigation in bioremediation and phytoremediation are discussed.     

Bacteria designed for bioremediation.

Although many environmental pollutants are efficiently degraded by microorganisms, others persist and constitute a severe health hazard. In some instances, persistence is a consequence of the inadequate catabolic potential of the available microorganisms. Gene technology, combined with a solid knowledge of catabolic pathways and microbial physiology, enables the experimental evolution of new or improved catabolic activities for such pollutants.     

Edible genetically modified microorganisms and plants for improved health

The development of new strategies for the delivery of vaccine antigens or immune modulators to the mucosal tissue includes innovative approaches such as the use of genetically modified food microorganisms and plants. Even though the 'proof-of-concept' has recently been established for these two systems, key questions mainly related to efficacy and risk of breaking oral tolerance remain to be critically addressed in the immediate future.     

Effect of natural and genetically modified rhizospheric Pseudomonas aureofaciens bacteria on accumulation of arsenic by plants

Gene constructions rendering bacteria resistant to arsenic and capable of dissolving phosphates and/or arsenates were created by cloning ars operon and the gene of citrate synthase from a chromosome of the strain Pseudomonas aeruginosa PAO1. Genetically modified variants of the strain Pseudomonas aureofaciens BS1393 have been constructed, which are resistant to high concentrations of arsenic and dissolve poorly soluble phosphates and/or arsenates. Recombinant strains P. aureofaciens BS1393(pUCP22::arsRBC) and P. aureofaciens BS1393(pUCP22::gltA) exerted positive effects on the survival of sorgo (Sorghum saccharatum L.) and its ability to accumulate arsenic.     

Novel roles for genetically modified plants in environmental protection

Transgenic plants of environmental benefit typically consist of plants that either reduce the input of agrochemicals into the environment or make the biological remediation of contaminated areas more efficient. Examples include the construction of species that result in reduced pesticide use and of species that contain genes for either the degradation of organics or the increased accumulation of inorganics. Cutting-edge approaches, illustrated by our own work, focus on the applicability of genetically modified (GM) plants that produce insect pheromones or that are specifically tailored to the phytoremediation of cadmium or PCBs. This paper discusses the role that the next generation of GM plants might play in preventing and reducing chemical contamination and in converting contaminated sites into safe agricultural or recreational land.     

Engineering bacteria for bioremediation

The treatment of environmental pollution by microorganisms is a promising technology. Various genetic approaches have been developed and used to optimize the enzymes, metabolic pathways and organisms relevant for biodegradation. New information on the metabolic routes and bottlenecks of degradation is still accumulating, enlarging the available toolbox. With molecular methods allowing the characterization of microbial community structure and activities, the performance of microorganisms under in situ conditions and in concert with the indigenous microflora will become predictable.     

Quantification of cuticular permeability in genetically modified plants

More and more studies on genetically modified plants are identifying parts of the genetic code with putative involvement in creating the cuticular barrier. Unfortunately, many of these studies suffer from the inadequacy of the chosen methods to quantify, in a reasonably unambiguous way, if and how the efficacy of the cuticular barrier is affected by the genetic change. A short overview of relevant findings is given and a more stringent experimental approach to quantifying effects on cuticular permeability in genetically modified plants proposed.     

Bioavailability of transgenic microRNAs in genetically modified plants

Background

Transgenic expression of small RNAs is a prevalent approach in agrobiotechnology for the global enhancement of plant foods. Meanwhile, emerging studies have, on the one hand, emphasized the potential of transgenic microRNAs (miRNAs) as novel dietary therapeutics and, on the other, suggested potential food safety issues if harmful miRNAs are absorbed and bioactive. For these reasons, it is necessary to evaluate the bioavailability of transgenic miRNAs in genetically modified crops.

Results

As a pilot study, two transgenic Arabidopsis lines ectopically expressing unique miRNAs were compared and contrasted with the plant bioavailable small RNA MIR2911 for digestive stability and serum bioavailability. The expression levels of these transgenic miRNAs in Arabidopsis were found to be comparable to that of MIR2911 in fresh tissues. Assays of digestive stability in vitro and in vivo suggested the transgenic miRNAs and MIR2911 had comparable resistance to degradation. Healthy mice consuming diets rich in Arabidopsis lines expressing these miRNAs displayed MIR2911 in the bloodstream but no detectable levels of the transgenic miRNAs.

Conclusions

These preliminary results imply digestive stability and high expression levels of miRNAs in plants do not readily equate to bioavailability. This initial work suggests novel engineering strategies be employed to enhance miRNA bioavailability when attempting to use transgenic foods as a delivery platform.

     

Risk from genetically engineered and modified marine fish

In support of the emerging industries of warmwater marine fish mariculture, genetic engineering and classical genetic improvement programmes have been initiated for a variety of exclusively marine fish. These programmes have the potential to perturb allele and genotype frequencies, or introduce novel alleles and genes into conspecific wild populations. Despite concerns to the contrary, the following hypothesis remains to be falsified: laboratory induced allele frequency/genotype changes and novel alleles or genes have a negligible probability of being selectively favoured in wild populations under natural selection, and accordingly, without sustained large scale releases, have little potential for ecological impact     

Review: genetically modified plants for the promotion of human health

Plants are attractive biological resources because of their ability to produce a huge variety of chemical compounds, and the familiarity of production in even the most rural settings. Genetic engineering gives plants additional characteristics and value for cultivation and post-harvest. Genetically modified (GM) plants of the “first generation” were conferred with traits beneficial to producers, whereas GM plants in subsequent “generations” are intended to provide beneficial traits for consumers. Golden Rice is a promising example of a GM plant in the second generation, and has overcome a number of obstacles for practical use. Furthermore, consumer-acceptable plants with health-promoting properties that are genetically modified using native genes are being developed. The emerging technology of metabolomics will also support the commercial realization of GM plants by providing comprehensive analyzes of plant biochemical components.     

Animal nutrition with feeds from genetically modified plants

Plant breeders have made and will continue to make important contributions toward meeting the need for more and better feed and food. The use of new techniques to modify the genetic makeup of plants to improve their properties has led to a new generation of crops, grains and their by-products for feed. The use of ingredients and products from genetically modified plants (GMP) in animal nutrition properly raises many questions and issues, such as the role of a nutritional assessment of the modified feed or feed additive as part of safety assessment, the possible influence of genetically modified (GM) products on animal health and product quality and the persistence of the recombinant DNA and of the 'novel' protein in the digestive tract and tissues of food-producing animals. During the last few years many studies have determined the nutrient value of GM feeds compared to their conventional counterparts and some have additionally followed the fate of DNA and novel protein. The results available to date are reassuring and reveal no significant differences in the safety and nutritional value of feedstuffs containing material derived from the so-called 1st generation of genetically modified plants (those with unchanged gross composition) in comparison with non-GM varieties. In addition, no residues of recombinant DNA or novel proteins have been found in any organ or tissue samples obtained from animals fed with GMP. These results indicate that for compositionally equivalent GMP routine-feeding studies with target species generally add little to nutritional and safety assessment. However, the strategies devised for the nutritional and safety assessment of the 1st generation products will be much more difficult to apply to 2nd generation GMP in which significant changes in constituents have been deliberately introduced (e.g., increased fatty acids or amino acids content or a reduced concentration of undesirable constituents). It is suggested that studies made with animals will play a much more important role in insuring the safety of these 2nd generation constructs.     

Tolerance to aluminum in genetically modified tobacco plants

Genetically modified tobacco plants tolerant to high aluminum concentrations were developed by integration of constructs containing rhamnolipid genes (rhlA and rhlB). At an aluminum concentration of 200 mM in ionite soil, the control plants perished, whereas the transgenic plants, although they were inhibited, continued to grow and produced seeds.     

The tolerance of tobacco genetically modified plants to aluminium

Introduction of the vector constructions with rhamnolipid genes (rhlA and rhlB) into tobacco plants has resulted in development of genetically modified plants which were tolerant to high concentrations of aluminium. While under aluminium concentration of 200 mM in a soil (ionit resin) all control plants perished, transgenic tobacco plants continued to grow and were fertile.     

Barriers to application of genetically modified lactic acid bacteria

To increase the acceptability of food products containing genetically modified microorganisms it is necessary to provide in an early stage to the consumers that the product is safe and that the product provide a clear benefit to the consumer. To comply with the first requirement a systematic approach to analyze the probability that genetically modified lactic acid bacteria will transform other inhabitants of the gastro-intestinal (G/I) tract or that these lactic acid bacteria will pick up genetic information of these inhabitants has been proposed and worked out to some degree. From this analysis it is clear that reliable data are still missing to carry out complete risk assessment. However, on the basis of present knowledge, lactic acid bacteria containing conjugative plasmids should be avoided. Various studies show that consumers in developed countries will accept these products when they offer to them health or taste benefits or a better keepability. For the developing countries the biggest challenge for scientists is most likely to make indigenous fermented food products with strongly improved microbiological stability due to broad spectra bacteriocins produced by lactic acid bacteria. Moreover, these lactic acid bacteria may contribute to health.     

Problem formulation in the environmental risk assessment for genetically modified plants

Problem formulation is the first step in environmental risk assessment (ERA) where policy goals, scope, assessment endpoints, and methodology are distilled to an explicitly stated problem and approach for analysis. The consistency and utility of ERAs for genetically modified (GM) plants can be improved through rigorous problem formulation (PF), producing an analysis plan that describes relevant exposure scenarios and the potential consequences of these scenarios. A properly executed PF assures the relevance of ERA outcomes for decision-making. Adopting a harmonized approach to problem formulation should bring about greater uniformity in the ERA process for GM plants among regulatory regimes globally. This paper is the product of an international expert group convened by the International Life Sciences Institute (ILSI) Research Foundation.     

Evolution of a regulatory framework for pharmaceuticals derived from genetically modified plants

The use of genetically modified (GM) plants to synthesize proteins that are subsequently processed, regulated and sold as pharmaceuticals challenges two very different established regulatory frameworks, one concerning GM plants and the other covering the development of biotechnology-derived drugs. Within these regulatory systems, specific regulations and guidelines for plant-made pharmaceuticals (PMPs) - also referred to as plant-derived pharmaceuticals (PDPs) - are still evolving. The products nearing commercial viability will ultimately help to road test and fine-tune these regulations, and might help to reduce regulatory uncertainties. In this review, we summarize the current state of regulations in different countries, discuss recent changes and highlight the need for further regulatory development in this burgeoning, new industry. We also make the case for the harmonization of international regulations.     

Assessing the risk of gene flow from genetically modified trees carrying mitigation transgenes

The increasing use of genetically modified (GM) plants has raised concerns about the escape of transgenes to conventional populations. To counteract possible fitness advantages of GM plants, an advantageous ‘primary transgene’ may be linked to a ‘mitigation’ transgene that is selectively disfavored. A risk related to this technique is the possible break-up of the transgenes. This may lead to the establishment of genotypes that only express the primary transgenic trait and are therefore selectively favored over both conventional and GM genotypes. We study here how the establishment of break-up genotypes in tree populations depends on life history, pollen dispersal kernels, and the linkage between transgenes. To this end, we model the dynamics of an initially homozygous GM population and a conventional population growing next to each other. The simulations are based on a spatially explicit simulation model for population-genotype dynamics. Pollen and seed dispersal are described by fat-tailed dispersal kernels. Break-up of transgenes is due subsequent crossing of GM and conventional genotypes. The spatial pattern of pollen and seed dispersal causes break-up and establishment of new invasive genotypes to occur mostly in the conventional population. The amount of individuals of these genotypes established in the conventional population depends on the interaction between life-history traits, pollen dispersal distances and the linkage of the transgenes. Thus, risk assessment of transgene break-up requires information on the genetics, local dynamics, and dispersal of GM and conventional varieties. The approach presented here provides a tool for combining these different sources of information, and it contributes to the formulation of general rules for the management of GM tree populations.