ReviewMultigene engineering: dawn of an exciting new era in biotechnology
Introduction
A vast majority of agronomic traits are quantitative and are controlled polygenetically. Genetic engineering is now moving from the initial phase of introducing single gene traits (e.g. resistance to herbicides, disease or insects) to multigenic traits [1], coding for complete metabolic pathways, bacterial operons or biopharmaceuticals that require an assembly of complex multisubunit proteins.
Multigene engineering via the nuclear genome involves several challenges. First, generation of transgenic lines expressing individual genes is necessary, because the nuclear genome does not process polycistrons. Second, such independent transgenic lines that harbor transgenes need to be brought together within a single host by repetitive breeding. Unfortunately, this step is complicated by gene silencing and position effects observed frequently in nuclear transgenic plants. Gene silencing has been observed because of the use of repetitive regulatory sequences, integration of multiple copies of the transgene or even as a result of the efficient transcription of transgenes; it occurs both at the transcriptional and post-transcriptional levels [2]. Position effects are caused by the random integration of transgenes into the nuclear genome. Screening of multiple transgenic lines might require the use of different selectable markers at each step. It is remarkable that, despite these technical hurdles, multiple genes have been skillfully engineered via the nuclear genome for the expression of vitamins 3••., 4.. However, these efforts have been highly time-consuming; for example, it took seven years to engineer three genes for the expression of provitamin A, even though the authors were fortunate to introduce two genes at once [5].
Fortunately, there are a few alternative approaches to overcome the aforementioned challenges. In one such effort, a series of three genes encoding a polyprotein containing three enzymes were introduced via the nuclear genome. The polyprotein consisted of tobacco vein mottling virus (TVMV) Nla proteinase and two other reporter genes— namely, acetate kinase and Tn9 chloramphenicol acetyl transferase—separated by the TVMV Nla proteinase recognition sequence. The Nla proteinase facilitated separation of the two enzymes, which were independently functional [6]. Although this approach has been used previously for multigene engineering, this study attempted to simultaneously express foreign proteins in the cytosol and chloroplasts. Such polyproteins should be modified, however, to ensure efficient and predictable processing of individual enzymes within chloroplasts.
Besides technical challenges in nuclear multigene engineering, there are unfortunate negative perceptions and environmental concerns about genetically modified food crops. Lack of gene containment owing to the pollen-mediated out-cross of transgenes from nuclear transgenic plants to related crops or weeds has been a major concern 5., 7.. In addition, the possibility of insects developing resistance to insecticidal proteins, due to low levels of transgene expression and toxicity of transgenic pollen to non-target insects, has raised environmental concerns for transgenic plants engineered for pest resistance 5., 7..
To address some of these environmental concerns and to facilitate multigene engineering in a single transformation step, the chloroplast genome has been targeted to express several foreign genes 8., 9.. Compartmentalization and expression of the transgenes in the maternally inherited chloroplasts should help to allay public concerns about gene containment 10., 11.. The ability of plants with transgenic chloroplasts to kill insects that developed very high levels of resistance (up to 40 000-fold) against Bacillus thuringiensis insecticidal proteins should also dispel the fear of insects developing resistance in the field [12]. Further, the lack of toxicity of transgenic pollen to non-target insects is yet another advantage of plants with transgenic chloroplasts [13••]. The capability of breaking expression level barriers without causing harmful effects to the host plant and the ability to engineer multiple genes in a single transformation event, are probably the greatest advantages of chloroplast genetic engineering. Coordinated expression of multiple genes, preferably driven by a single promoter, is especially important for stoichiometric synthesis and assembly of multisubunit proteins like monoclonal antibodies [14]. Observations of nearly 50% foreign protein in the total soluble protein (tsp) [13••] and 17 000% more transcripts in chloroplast transgenic plants than nuclear transgenic plants [15•] assuages the concerns of gene silencing at the transcriptional or post-transcriptional level. Position effects are not observed in chloroplast genetic engineering because of targeted gene integration; several independent chloroplast transgenic lines express foreign proteins to the same level, except for minor physiological variations [16•]. In some cases, manipulation of a pathway or hyper-expression of a transgene is very demanding on nuclear transgenic plants, resulting in deleterious pleiotropic effects including stunted growth and sterility. However, such pleiotropic effects observed in nuclear transgenic plants were alleviated when the same foreign proteins were compartmentalized within transgenic chloroplasts 15•., 16•., 17.. Other recent developments in chloroplast genetic engineering have been the advent of a plant-derived selectable marker [18••] and transformation of the chloroplast genome of edible plant species, including potato and tomato 19., 20•.. This review discusses recent achievements and forecasts the future role of chloroplast and nuclear transformation in multigene engineering of plants.
Section snippets
Nuclear multigene engineering
A significant recent step in multigene engineering has been the development of a rice variety that accumulates provitamin A [3••]. Vitamin A deficiency results in various diseases like night-blindness or even complete blindness. It is estimated that improved vitamin A nutrition can help to prevent over one to two million deaths each year among children aged one to four years. Employing Agrobacterium-mediated transformation, three genes essential for the synthesis of the enzymes of the
Chloroplast multigene engineering
The concept of chloroplast transformation, conceived in the mid-80s 23., 24., has recently blossomed into a safe and environmentally friendly technology 8., 9., 25.. When the first transgenes were introduced via the chloroplast genome, it was believed that foreign genes could be inserted only into transcriptionally silent spacer regions, amidst divergent chloroplast genes [26]. However, Daniell et al. [10] advanced the concept of inserting transgenes into functional operons and
Conclusions
Plant biotechnology is at the threshold of an exciting new era in which the emphasis is on the introduction of traits that require the manipulation of metabolic pathways or coordinated expression of multisubunit proteins. The development of rice varieties enriched in provitamin A is an early success story in this new era. The chloroplast transgenic approach has facilitated expression of bacterial operons and biopharmaceuticals at unprecedented levels, never before reported in the literature.
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
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