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Plant Biotechnology: From basic science to industrial applications
J. Plant Physiol. 160. 723 – 725 (2003) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Plant Biotechnology: From basic science to industrial applications Uwe Sonnewald* Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, 06466 Gatersleben, Germany
Due to tremendous achievements made in all disciplines of plant sciences enormous opportunities to improve performance and quality of crop plants are awaiting industrial exploitation. However, the opinion on utility and risk of genetically modified (GM) crops is sharply divided. Some argue that GMs are necessary, entirely safe and essential to feed a growing world population, others state they are not needed, are inherently unsafe, and hold great risks. In this issue different aspects of plant biotechnology are discussed in nine overview articles. The main focus is on metabolic engineering of biosynthetic pathways enabling the production of renewable resources and valuable biomolecules in crop plant. In addition possible reasons for the uneven development of plant biotechnology between Europe and the USA and future perspectives of commercialisation of GM crops are discussed. In his article (pp. 727–734) R. Emrich summarizes industrial expectations and the potential benefits of plant biotechnology. Starting with the first transgenic tobacco plants in 1983, genetic engineering has become a more or less routine technology in basic science and plant biotechnology. The invention of marker genes conferring antibiotic or herbicide resistance enabled the efficient selection of transformed cells. In the United States delayed-ripening tomatoes (FlavrSavrTM) came on to the market in 1994 and were the first genetically modified food available to consumers. Since then the global area of transgenic crops continuously grew. The area planted to GM crops shot up from 1.7 million hectares in 1996 to 58.7 in 2002 (source: http://www.isaaa.org). In 2002, four countries accounted for 99 % of the global genetically modified crop area. The United States grew 39.0 million hectares (66.4 % of global total), followed by Argentina with 13.5 million hectares (23 %), Canada 3.5 million hectares (6 %) and China 2.1 million hectares (3.6 %). Amongst the different crops, soybean, cotton, canola and maize are the four principal crops in which transgenic technology is utilized. Herbi* E-mail: [email protected]
cide tolerant soybean is the dominant transgenic crop commercially grown, representing 62 % of the global transgenic crop area in 2002. The second most dominant crop is Bt maize, which occupied 7.7 million hectares in 2002. The third most dominant crop is herbicide tolerant canola, which was grown on 3.0 million hectares, equivalent to 5 % of global transgenic area in 2002. This triumphal march of genetically modified crop plants in Northern America has not been continued in the EU member states. This is mainly due to limited public acceptance and unclear administrative legislation. An overview concerning the current status of genetically modified plants in Europe and the United States of America is given by P. Brandt (pp. 735–742). The first generation of transgenic crop plants is characterized by the introduction of single target genes commonly associated with an antibiotic or herbicide resistance gene. Due to consumer concerns the use of most antibiotic resistance genes will probably no longer be acceptable and an increasing number of genetically modified plants resistant to herbicides might ultimately lead to the accelerated appearance of resistant weeds rendering the use of the respective chemical inefficient. An additional limitation of todays GMs is that plant transformation leads to random integration of transgenes into the plant genome. This on the one hand results in unpredictable expression levels and furthermore, insertional mutagenesis cannot be excluded. To circumvent these problems and to improve consumer acceptance marker-free GMPs which harbour the transgene of interest at a defined genomic location would be beneficial. Therefore, several attempts to achieve site-specific integration of transgenes have been undertaken. However, to date no reliable and efficient system has been reported which would be applicable on an industrial scale. Latest developments in homologous recombination and marker gene excision are discussed by H. Puchta (pp. 743–754). Beside insect and herbicide resistance, crop plants have been engineered to cope with adverse environmental conditions such as drought, cold and high salt or have 0176-1617/03/160/07-723 $ 15.00/0
724
Uwe Sonnewald
been modified with respect to altered fruit ripening, cell wall turnover, oil or starch composition, vitamin content and the production of industrial or pharmaceutical biomolecules. Qualitative improvements of crop plants can be achieved by molecular farming and metabolic engineering. Molecular farming refers to the production of valuable polypeptides in GMPs. Plants represent an attractive and cost-efficient alternative to conventional systems for the production of biomolecules. Amongst others, obvious advantages are the low energy costs for production, the flexibility of production, the possibility to produce bulk quantities, the occurrence of eucaryotic posttranslational protein modifications and the absence of human pathogens or toxins. Benefits and risks of the production of pharmaceutical proteins in transgenic plants are discussed by Warzecha and Mason (pp. 755–764). Metabolic engineering describes the modulation of metabolic and biosynthetic networks of an organism with the intention to direct metabolic flux into the biochemical pathway of a certain valuable molecule. In plants metabolic engineering is based on the understanding of numerous biochemical and physiological mechanisms which have to be considered. Amongst these, availability of precursors, compartmentation (cellular and sub-cellular) of enzymes and substrates, flux into competing pathways, overall regulation of the biochemical pathways (metabolic network) and degradation and storage of the product are most important. Thus, a prerequisite for effective metabolic engineering is sufficient biochemical knowledge concerning the control of the metabolic pathway under study. In addition, manipulations should be as targeted as possible. Plants as sedentary organisms have evolved a multitude of compounds protecting them against predators, abiotic and biotic stresses. Therefore, metabolic engineering approaches should not affect pathways leading to the production of essential components required for stress adaptation or normal plant development. Furthermore, crop plants have been optimised for high yield. As yield represents the highest value of any crop plant this parameter should in general not be negatively influenced. One of the most extensively studied example of metabolic engineering is the manipulation of starch quality and quantity. Starch represents an important reserve carbohydrate in most plants and is used for many industrial applications. Depending on its composition starch possesses different physicochemical properties suitable for diverse applications in the paper-, textile-, plastics-, food- and pharmaceutical industry. Since natural starches are composed of two different polymers, amylose and amylopectin, both with different physicochemical properties, research has been directed towards the creation of uniform and novel polymers. Attempts to modify starch composition and possible applications of the engineered starch are discussed by G. A. Kok-Jacon et al. (pp. 765–777). Starch is not the only plant product which has successfully been manipulated. Amongst others, fatty acid composition and fructan accumulation have been targeted by genetic engineering. Fats and oils are stored in form of tria-
cylglycerides, in which the fatty acids are linked by ester bonds to the three hydroxyl groups of glycerol. Carbon chain length can vary from 8 to 20 but most commonly 16 and 18 carbon fatty acids are found in higher plants. More than 200 types of fatty acids are known to be produced in plants, but only a few of them (palmitic, stearic, oleic, linoleic and linolenic acids) are commercially exploited. Approximately 90 % of vegetable oil produced is used for human consumption whereas only 10 % is used for non-food applications. Ongoing attempts to engineer fatty acids for improved nutritional characteristics are discussed by H. Drexler et al. (pp. 779 – 802). Fructans are polymers of fructose carrying a D-glucosyl residue at the end of the chain attached via a β-2,1-linkage. Plant fructans can be classified in three main types, inulin, phlein and branched inulin. They represent storage carbohydrates in several plant species including grasses. Fructans are supposed to be beneficial for human health by promoting growth of beneficial Lactobacilli and Bifidobacteria. At present Chicory inulin is the primary fructan used as food ingredient. Besides plants, many microrganism produce fructans, however, chain length of bacterial fructans is much larger than in plants. Due to health promoting properties of fructans considerable interest to create transgenic crop plants accumulating high levels of fructans exists (summarized in Ritsema and Smeekens, pp. 811– 820). First attempts to engineer metabolic pathways in transgenic plants concentrated on starch, fatty acids and amino acid biosynthesis. This was mainly due to existing biochemical knowledge, the occurrence of mutants and the availability of genes involved. However, plants are also the major source of vitamins in the human diet. Therefore, attempts have been undertaken to improve vitamin content in plants. Most well known examples are transgenic plants with elevated levels of provitamin A («Golden rice»), vitamin C and E. Due to limited knowledge concerning biosynthesis and degradation of most vitamins, only moderate increases in the content of some vitamins in transgenic plants have been reported. The current knowledge in the field is discussed by K. Herbers (pp. 821– 829). In addition to the manipulation of endogenous plant products discussed above, attempts to redirect carbon flow towards the production of novel biomolecules have been reported. One of the first examples for production of a non-plant biopolymer in transgenic plants was the synthesis of polyhydroxyalkanoates (PHA). PHAs are polyesters of hydroxyacids synthesised in many bacteria as storage compound. Since PHAs are biodegradable and biocompatible plastics they are used in medical applications, such as implants and gazes. Due to high costs for production of bacterial PHAs their use is restricted to medical applications and the substitution of plastics for packaging material and disposable items is not feasible. Therefore, transgenic plants could offer an attractive alternative for the cost effective production of these biopolymers. L. Moire et al. (pp. 831– 839) discuss the use of transgenic plants as bioreactors for the synthesis of novel biopolymers.
Plant Biotechnology: From basic science to industrial applications
725
References Biesgen C, Herbers K (2000) Phytofarming approaches to foreign protein expression in higher plants. J Plant Biotechnol 2: 1–12 Chen TH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5: 250 – 257 Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6: 219 – 226
Doran PM (2000) Foreign protein production in plant tissue cultures. Curr. Opin. Biotechnol 11: 199 – 204 Herbers K, Sonnewald U (1999) Production of new/modified proteins in transgenic plants. Curr Opin Biotechnol 10: 163–168 Sonnewald U, Herbers K (2001) Plant Biotechnology – Methods, Goals and Achievements. In: Nösberger J, Geiger HH, Struik PC, eds., Crop Science – Progress and Prospects, CABI Publishing, Wallingford: 329 – 350
Plant Biotechnology: From basic science to industrial applications Uwe Sonnewald* Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 3, 06466 Gatersleben, Germany
Due to tremendous achievements made in all disciplines of plant sciences enormous opportunities to improve performance and quality of crop plants are awaiting industrial exploitation. However, the opinion on utility and risk of genetically modified (GM) crops is sharply divided. Some argue that GMs are necessary, entirely safe and essential to feed a growing world population, others state they are not needed, are inherently unsafe, and hold great risks. In this issue different aspects of plant biotechnology are discussed in nine overview articles. The main focus is on metabolic engineering of biosynthetic pathways enabling the production of renewable resources and valuable biomolecules in crop plant. In addition possible reasons for the uneven development of plant biotechnology between Europe and the USA and future perspectives of commercialisation of GM crops are discussed. In his article (pp. 727–734) R. Emrich summarizes industrial expectations and the potential benefits of plant biotechnology. Starting with the first transgenic tobacco plants in 1983, genetic engineering has become a more or less routine technology in basic science and plant biotechnology. The invention of marker genes conferring antibiotic or herbicide resistance enabled the efficient selection of transformed cells. In the United States delayed-ripening tomatoes (FlavrSavrTM) came on to the market in 1994 and were the first genetically modified food available to consumers. Since then the global area of transgenic crops continuously grew. The area planted to GM crops shot up from 1.7 million hectares in 1996 to 58.7 in 2002 (source: http://www.isaaa.org). In 2002, four countries accounted for 99 % of the global genetically modified crop area. The United States grew 39.0 million hectares (66.4 % of global total), followed by Argentina with 13.5 million hectares (23 %), Canada 3.5 million hectares (6 %) and China 2.1 million hectares (3.6 %). Amongst the different crops, soybean, cotton, canola and maize are the four principal crops in which transgenic technology is utilized. Herbi* E-mail: [email protected]
cide tolerant soybean is the dominant transgenic crop commercially grown, representing 62 % of the global transgenic crop area in 2002. The second most dominant crop is Bt maize, which occupied 7.7 million hectares in 2002. The third most dominant crop is herbicide tolerant canola, which was grown on 3.0 million hectares, equivalent to 5 % of global transgenic area in 2002. This triumphal march of genetically modified crop plants in Northern America has not been continued in the EU member states. This is mainly due to limited public acceptance and unclear administrative legislation. An overview concerning the current status of genetically modified plants in Europe and the United States of America is given by P. Brandt (pp. 735–742). The first generation of transgenic crop plants is characterized by the introduction of single target genes commonly associated with an antibiotic or herbicide resistance gene. Due to consumer concerns the use of most antibiotic resistance genes will probably no longer be acceptable and an increasing number of genetically modified plants resistant to herbicides might ultimately lead to the accelerated appearance of resistant weeds rendering the use of the respective chemical inefficient. An additional limitation of todays GMs is that plant transformation leads to random integration of transgenes into the plant genome. This on the one hand results in unpredictable expression levels and furthermore, insertional mutagenesis cannot be excluded. To circumvent these problems and to improve consumer acceptance marker-free GMPs which harbour the transgene of interest at a defined genomic location would be beneficial. Therefore, several attempts to achieve site-specific integration of transgenes have been undertaken. However, to date no reliable and efficient system has been reported which would be applicable on an industrial scale. Latest developments in homologous recombination and marker gene excision are discussed by H. Puchta (pp. 743–754). Beside insect and herbicide resistance, crop plants have been engineered to cope with adverse environmental conditions such as drought, cold and high salt or have 0176-1617/03/160/07-723 $ 15.00/0
724
Uwe Sonnewald
been modified with respect to altered fruit ripening, cell wall turnover, oil or starch composition, vitamin content and the production of industrial or pharmaceutical biomolecules. Qualitative improvements of crop plants can be achieved by molecular farming and metabolic engineering. Molecular farming refers to the production of valuable polypeptides in GMPs. Plants represent an attractive and cost-efficient alternative to conventional systems for the production of biomolecules. Amongst others, obvious advantages are the low energy costs for production, the flexibility of production, the possibility to produce bulk quantities, the occurrence of eucaryotic posttranslational protein modifications and the absence of human pathogens or toxins. Benefits and risks of the production of pharmaceutical proteins in transgenic plants are discussed by Warzecha and Mason (pp. 755–764). Metabolic engineering describes the modulation of metabolic and biosynthetic networks of an organism with the intention to direct metabolic flux into the biochemical pathway of a certain valuable molecule. In plants metabolic engineering is based on the understanding of numerous biochemical and physiological mechanisms which have to be considered. Amongst these, availability of precursors, compartmentation (cellular and sub-cellular) of enzymes and substrates, flux into competing pathways, overall regulation of the biochemical pathways (metabolic network) and degradation and storage of the product are most important. Thus, a prerequisite for effective metabolic engineering is sufficient biochemical knowledge concerning the control of the metabolic pathway under study. In addition, manipulations should be as targeted as possible. Plants as sedentary organisms have evolved a multitude of compounds protecting them against predators, abiotic and biotic stresses. Therefore, metabolic engineering approaches should not affect pathways leading to the production of essential components required for stress adaptation or normal plant development. Furthermore, crop plants have been optimised for high yield. As yield represents the highest value of any crop plant this parameter should in general not be negatively influenced. One of the most extensively studied example of metabolic engineering is the manipulation of starch quality and quantity. Starch represents an important reserve carbohydrate in most plants and is used for many industrial applications. Depending on its composition starch possesses different physicochemical properties suitable for diverse applications in the paper-, textile-, plastics-, food- and pharmaceutical industry. Since natural starches are composed of two different polymers, amylose and amylopectin, both with different physicochemical properties, research has been directed towards the creation of uniform and novel polymers. Attempts to modify starch composition and possible applications of the engineered starch are discussed by G. A. Kok-Jacon et al. (pp. 765–777). Starch is not the only plant product which has successfully been manipulated. Amongst others, fatty acid composition and fructan accumulation have been targeted by genetic engineering. Fats and oils are stored in form of tria-
cylglycerides, in which the fatty acids are linked by ester bonds to the three hydroxyl groups of glycerol. Carbon chain length can vary from 8 to 20 but most commonly 16 and 18 carbon fatty acids are found in higher plants. More than 200 types of fatty acids are known to be produced in plants, but only a few of them (palmitic, stearic, oleic, linoleic and linolenic acids) are commercially exploited. Approximately 90 % of vegetable oil produced is used for human consumption whereas only 10 % is used for non-food applications. Ongoing attempts to engineer fatty acids for improved nutritional characteristics are discussed by H. Drexler et al. (pp. 779 – 802). Fructans are polymers of fructose carrying a D-glucosyl residue at the end of the chain attached via a β-2,1-linkage. Plant fructans can be classified in three main types, inulin, phlein and branched inulin. They represent storage carbohydrates in several plant species including grasses. Fructans are supposed to be beneficial for human health by promoting growth of beneficial Lactobacilli and Bifidobacteria. At present Chicory inulin is the primary fructan used as food ingredient. Besides plants, many microrganism produce fructans, however, chain length of bacterial fructans is much larger than in plants. Due to health promoting properties of fructans considerable interest to create transgenic crop plants accumulating high levels of fructans exists (summarized in Ritsema and Smeekens, pp. 811– 820). First attempts to engineer metabolic pathways in transgenic plants concentrated on starch, fatty acids and amino acid biosynthesis. This was mainly due to existing biochemical knowledge, the occurrence of mutants and the availability of genes involved. However, plants are also the major source of vitamins in the human diet. Therefore, attempts have been undertaken to improve vitamin content in plants. Most well known examples are transgenic plants with elevated levels of provitamin A («Golden rice»), vitamin C and E. Due to limited knowledge concerning biosynthesis and degradation of most vitamins, only moderate increases in the content of some vitamins in transgenic plants have been reported. The current knowledge in the field is discussed by K. Herbers (pp. 821– 829). In addition to the manipulation of endogenous plant products discussed above, attempts to redirect carbon flow towards the production of novel biomolecules have been reported. One of the first examples for production of a non-plant biopolymer in transgenic plants was the synthesis of polyhydroxyalkanoates (PHA). PHAs are polyesters of hydroxyacids synthesised in many bacteria as storage compound. Since PHAs are biodegradable and biocompatible plastics they are used in medical applications, such as implants and gazes. Due to high costs for production of bacterial PHAs their use is restricted to medical applications and the substitution of plastics for packaging material and disposable items is not feasible. Therefore, transgenic plants could offer an attractive alternative for the cost effective production of these biopolymers. L. Moire et al. (pp. 831– 839) discuss the use of transgenic plants as bioreactors for the synthesis of novel biopolymers.
Plant Biotechnology: From basic science to industrial applications
725
References Biesgen C, Herbers K (2000) Phytofarming approaches to foreign protein expression in higher plants. J Plant Biotechnol 2: 1–12 Chen TH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5: 250 – 257 Daniell H, Streatfield SJ, Wycoff K (2001) Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 6: 219 – 226
Doran PM (2000) Foreign protein production in plant tissue cultures. Curr. Opin. Biotechnol 11: 199 – 204 Herbers K, Sonnewald U (1999) Production of new/modified proteins in transgenic plants. Curr Opin Biotechnol 10: 163–168 Sonnewald U, Herbers K (2001) Plant Biotechnology – Methods, Goals and Achievements. In: Nösberger J, Geiger HH, Struik PC, eds., Crop Science – Progress and Prospects, CABI Publishing, Wallingford: 329 – 350