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Plant genetic engineering to improve biomass characteristics for biofuels.

Currently, most ethanol produced in the United States is derived from maize kernel, at levels in excess of four billion gallons per year. Plant lignocellulosic biomass is renewable, cheap and globally available at 10-50 billion tons per year. At present, plant biomass is converted to fermentable sugars for the production of biofuels using pretreatment processes that disrupt the lignocellulose and remove the lignin, thus allowing the access of microbial enzymes for cellulose deconstruction. Both the pretreatments and the production of enzymes in microbial tanks are expensive. Recent advances in plant genetic engineering could reduce biomass conversion costs by developing crop varieties with less lignin, crops that self-produce cellulase enzymes for cellulose degradation and ligninase enzymes for lignin degradation, or plants that have increased cellulose or an overall biomass yield.

Biomass↗

Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures.

The production of the blue dye indigo in plants has been assumed to be a possible route to the introduction of novel coloration into flowers or fibres. As the human cytochrome P450 mono-oxygenase 2A6 (CYP2A6) can form indigo in bacterial cultures, we investigated whether the expression of the corresponding cDNA in transgenic plants could lead to indigo formation. In a first attempt, we generated tobacco cell suspension cultures expressing the cDNA encoding human CYP2A6. Supplementation of the medium with indole led to the generation of indican (3-hydroxyindole-beta-d-glucoside), a metabolite usually exclusively present in indigoferous dye plants. Hence, the recombinant CYP2A6 converted indole to the reactive metabolite 3-hydroxyindole (indoxyl), whereas rapid glucosylation is obviously conducted by ubiquitous plant glucosyl transferases (GTs). Interestingly, of nine additionally tested plant cell suspension cultures from various plant families, five were also capable of the formation of indican after indole supplementation, although this metabolism was more pronounced in transgenic tobacco cell suspension cultures expressing CYP2A6 cDNA. To evaluate whether indican or even indigo could be produced in whole plants, we generated transgenic tobacco plants harbouring active CYP2A6 together with an indole synthase (BX1) from maize. The genetically engineered tobacco plants accumulated indican, but did not develop a blue coloration. Although the de novo formation of indican in transgenic tobacco plants hampered indigo formation, it supports the contention that biosynthetic pathways can be efficiently mimicked by metabolic engineering.

Aryl Hydrocarbon Hydroxylases↗

Metabolic engineering of plant alkaloid biosynthesis.

Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Alkaloids↗

Genetic engineering of plants for virus resistance.

Historically, control of plant virus disease has involved numerous strategies which have often been combined to provide effective durable resistance in the field. In recent years, the dramatic advances obtained in plant molecular virology have enhanced our understanding of viral genome organizations and gene functions. Moreover, genetic engineering of plants for virus resistance has recently provided promising additional strategies for control of virus disease. At present, the most promising of these has been the expression of coat-protein coding sequences in plants transformed with a coat protein gene. Other potential methods include the expression of anti-sense viral transcripts in transgenic plants, the application of artificial anti-sense mediated gene regulation to viral systems, and the expression of viral satellite RNAs, RNAs with endoribonuclease activity, antiviral antibody genes, or human interferon genes in plants.

Base Sequence↗

Strategies to improve plant resistance to bacterial diseases through genetic engineering.

Many different genetic strategies have been proposed to engineer plant resistance to bacterial diseases, including producing antibacterial proteins of non-plant origin, inhibiting bacterial pathogenicity or virulence factors, enhancing natural plant defenses and artificially inducing programmed cell death at the site of infection. These are based on our knowledge of the mechanisms of action of antibacterial compounds and of the successive steps in plant-bacterial interactions. This article presents the different approaches and demonstrates that, even though several of these ideas have already been applied, no commercial applications have yet been achieved.

Animals↗

[Plant genetic engineering in Monsanto company: from the first laboratory experiments to worldwide practical use].

The history of modern biotechnology of agricultural plants is briefly considered in the article. Methods of genetic transformation and regeneration of transgenic plants as well as the mechanisms of resistance of genetically modified plants to herbicides and pests are discussed. By the example of genetically modified varieties and hybrids there are shown the ways of solving the problem of weeds and pests. The questions of biosafety legislation in different countries are considered.

Biotechnology↗

Unstable liaisons: the use of transposons in plant genetic engineering.

Transposable elements can be used to activate gene expression in clonal populations of cells and consequently to generate genetic mosaics. Plant mosaics, having organs composed of intermixed tissues with wild-type and mutant appearance, can be used to evaluate whether the action of a morphogenetic gene is cell-autonomous. Transposon-split gene constructions could also be used to raise plants transgenic for lethal genes and, in more general terms, they could be an ancillary tool in several types of biological experiment.

DNA Transposable Elements↗

Manipulating gene expression for the metabolic engineering of plants.

Introducing and expressing foreign genes in plants present many technical challenges that are not encountered with microbial systems. This review addresses the variety of issues that must be considered and the variety of options that are available, in terms of choosing transformation systems and designing recombinant transgenes to ensure appropriate expression in plant cells. Tissue specificity and proper developmental regulation, as well as proper subcellular localization of products, must be dealt with for successful metabolic engineering in plants..

Biomarkers↗

Site-specific recombination for genetic engineering in plants.

Site-specific recombination has been developed into a genetic engineering tool for higher eukaryotes. The manipulation of newly introduced DNA is now possible in the course of genetic transformation procedures, thus making the process more predictable and reliable. Also, a wide variety of chromosomal rearrangements using site-specific recombination have been documented both in metazoan and plant species. Applying such methods to plants opens new avenues for large-scale chromosome engineering in the future.

Chromosomes, Plant↗

Engineering the plant cell factory for secondary metabolite production.

Plant secondary metabolism is very important for traits such as flower color, flavor of food, and resistance against pests and diseases. Moreover, it is the source of many fine chemicals such as drugs, dyes, flavors, and fragrances. It is thus of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a fine chemical, to produce less of a toxic compound, or even to make new compounds, Engineering of plant secondary metabolism is feasible nowadays, but it requires knowledge of the biosynthetic pathways involved. To increase secondary metabolite production different strategies can be followed, such as overcoming rate limiting steps, reducing flux through competitive pathways, reducing catabolism and overexpression of regulatory genes. For this purpose genes of plant origin can be overexpressed, but also microbial genes have been used successfully. Overexpression of plant genes in microorganisms is another approach, which might be of interest for bioconversion of readily available precursors into valuable fine chemicals. Several examples will be given to illustrate these various approaches. The constraints of metabolic engineering of the plant cell factory will also be discussed. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks.

Bioreactors↗

Metabolic engineering of plant secondary products.

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.

Gene Expression↗

Metabolic engineering of plants for osmotic stress resistance.

Genes encoding critical steps in the synthesis of osmoprotectant compounds are now being expressed in transgenic plants. These plants generally accumulate low levels of osmoprotectants and have increased stress tolerance. The next priority is therefore to engineer greater osmoprotectant synthesis without detriment to the rest of metabolism. This will require manipulation of multiple genes, guided by thorough analysis of metabolite fluxes and pool sizes.

Betaine↗

Bioreactor technology: a novel industrial tool for high-tech production of bioactive molecules and biopharmaceuticals from plant roots.

Plants are the richest source for different bioactive molecules. Because of the vast number of side effects associated with synthetic pharmaceuticals, medical biotechnologists turned to nature to provide new promising therapeutic molecules from plant biofactories. The large-scale availability of the disease- and pesticide-free raw material is, however, restricted in vivo. Many bioactive plant secondary metabolites are accumulated in roots. Engineered plants can also produce human therapeutic proteins. Vaccines and diagnostic monoclonal antibodies can be won from their roots, so that engineered plants hold immense potential for the biopharmaceutical industry. To obtain sufficient amounts of the plant bioactive molecules for application in human therapy, adventitious and hairy roots have to be cultured in in vitro systems. High-tech pilot-scale bioreactor technology for the establishment of a long-term adventitious root culture from biopharmaceutical plants has recently been established. In this review, I briefly discuss a technology for cultivating bioactive molecule-rich adventitious and hairy roots from plants using a high-tech bioreactor system, as well as the principles and application of genome-restructuring mechanisms for plant-based biopharmaceutical production from roots. High-tech bioreactor-derived bioactive phytomolecules and biopharmaceuticals hold the prospect of providing permanent remedies for improving human well-being.

Biopharmaceutics↗