Although the enzymological sequences and intermediates of many metabolic pathways in a small number of well-studied organisms are known with some confidence, little is known in quantitative terms about the controls and integration of these pathways.  The necessary knowledge also includes conceptual and technical approaches necessary to understand the integration and control of genetic, catalytic, and transport processes.  Though there are notable exceptions, most successful attempts at metabolic engineering thus far have focused on modifying (positively or negatively) the expression of single genes (or a series of individual enzymatic steps) affecting pathways.  Generally, more success has been achieved when conversion or modification of an existing compound to another has been targeted than when an attempt has been made to significantly change flux through a pathway

Metabolic engineering is generally defined as the redirection of one or more enzymatic reactions to improve the production of existing compounds, produce new compounds or mediate the degradation of compounds. Substrate-product relationships in plant pathways were initially elucidated through the application of radiolabel tracer studies during the 1960s and 1970s. In the 1980s, with the advent of recombinant DNA technology tools were developed such as cloning, promoter analysis, protein targeting, plant transformation and biochemical genetics which facilitated the application of this knowledge to engineer metabolic pathways. Significant progress has been made in recent years in the molecular dissection of many plant pathways and the use of cloned genes to engineer plant metabolism. Although there are numerous success stories, there has been an even greater number of studies that have yielded completely unanticipated results. Such data underscore the fragmented state of our understanding of plant metabolism and highlight the growing gap between our ability to clone, study and manipulate individual genes and proteins and our understanding of how they are integrated into and impact the complex metabolic networks in plants. (della penna, 2001)

Macro: Protein

Although progress in pathway gene discovery and our ability to manipulate gene expression in transgenic plants has been most impressive during the past two decades, attempts to use these tools to engineer plant metabolism has met with more limited success. Though there are notable exceptions, most attempts at metabolic engineering have focused on modifying (positively or negatively) the expression of single genes (or series of individual enzymatic steps) affecting pathways. On the whole the predictability of success has been much better when one is targeting conversion or modification of an existing compound to another rather than attempting to increase flux through a pathway. Modifications to metabolic storage products or secondary metabolic pathways, which often have relatively flexible roles in plant biology, have also been generally more successful than manipulations of primary and intermediary metabolism (Della Penna, 1999).

At the macro- level most plants are not complete sources of all essential amino acids. The cereals, maize, wheat rice etc. tend to be low in lysine, while legumes are often low in sulfur rich amino acids such as methionine and cysteine. New maize and soybean varieties that contain higher levels of the amino acids lysine, are expected soon.  Consumption of foods made from these crops can help to prevent malnutrition in developing countries, plus prevent childhood blindness caused by lysine deficiency.

One could modify storage protein composition by introducing heterologous or homologous genes containing elevated levels of sulphur containing amino acids (methionine, cysteine) and lysine.  An 11kDa synthetic protein, MB1, was created to contain the maximum number of the essential amino acids methionine, threonine, lysine and leucine in a stable helical conformation; the structure was also designed to resist proteases (Beauregard et al. 1995). The physical properties, 16% methionine and 12% lysine content, make it a desirable candidate for improving soy protein quality. This protein was designed for expression in rumen bacteria, however, so it was necessary to ensure that it is stable in plant cells before attempting to engineer expression in soybean seed. It was subsequently targeted to seed protein storage bodies using appropriate leader sequences and seed specific promoters in transformation vectors (Simmonds and Donaldson 1999). Transgenic plants of commercial cultivars may be screened for their performance in soy food products, while transgenic plants of the anti-nutrient negative (null BBTI) lines should be superior material for animal feed

Poultry and swine can only absorb amino acids from their feed rations in highly specific ratios.  Those animals metabolize and excrete in the form of nitrogen pollution the amino acids that are caused to be “in excess” by a shortfall in the primary amino acids required in those animal-nutrition ratios.  The primary requirements for maize/soymeal-based feed rations are usually lysine and methionine.  High-lysine & high-methionine maize and soybeans could allow feed ration formulations that reduce animal nitrogen excretion by providing an improved balance of essential amino acids.  That can be accomplished now, but only by adding costly synthetic lysine and methionine to the feed ration.

Pioneer Hi-Bred International wishing to improve the amino acid ratio of soybeans for animal feed by introducing a gene from Brazil nut which coded for the sulphur rich protein albumin. This protein happens to be the very component of nuts that constitutes the greatest source of allergenicity.  Before release of the product, during consultation with the FDA, Pioneer Hi-Bred identified the allergen. Confronted with potential product liability and the costs of labeling all products derived from the new plant variety, the company abandoned plans to use the new soybeans in consumer products. No consumers were exposed to injury.

This effect soured the notion of using nuts as a source of sulphur-rich proteins. An Indian researcher looked to amaranthus to hunt out the proteins with the most desirable traits. Potato is the most important noncereal food crop and ranks fourth in terms of total global food production, besides being used as animal feed and as raw material for manufacture of starch, alcohol, and other food products. The essential amino acids that limit the nutritive value of potato protein are lysine, tyrosine, and the sulfur-containing amino acids methionine and cysteine. Chakraborty et al (2000) have reported introducing a gene into potato that is nonallergenic in nature and is rich in all essential amino acids, and the composition corresponds well with the World Health Organization standards for optimal human nutrition. There was a striking increase in the growth and production of tubers in transgenic populations and also of the total protein content with an increase in most essential amino acids (Chakraborty, 2000). The results document, in addition to successful nutritional improvement of potato tubers, the feasibility of genetically modifying other crop plants with novel seed protein composition.

Sweet potato is a crucial component of the diet in many developing countries such as Africa and South East Asia and is especially important in children’s diets.  Work being conducted by Prakash at Tuskegee University is focusing on many aspects of improving characteristics of this crop including disease resistance but of special interest is that work on improving the protein quality. In a study funded by NASA, they have engineered sweet potato plants with an artificial storage protein (ASP-1) gene. Transgenic plants exhibited a two and five fold increase in the total protein content in leaves and roots respectively over that of the control. A significant increase in the level of essential amino acids such as methionine, threonine, tryptophan, isoleucine, and lysine was also observed along with an increase in total nitrogen content. This has tremendous potential to positively impact the health and nutrition of these people.

Some proteins affect other aspects of food functionality such as for example baking quality of wheat.  The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain; the former being related to differences in the number of expressed HMW subunit genes. Barro et al (1998) transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour. A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits. Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering.

 However in metabolic engineering, one needs to be aware of the issue of flux. When faced with the novel experimental possibilities that molecular, genomic, and transgenic approaches have presented over the past two decades, researchers can be tempted to become fixated on producing transgenic plants and lose appreciation for the important roles enzyme kinetics play at individual reaction steps and within entire pathways.  Attempts to manipulate the lysine content of seeds illustrate that one needs to consider catabolic, as well as anabolic, variables when trying to engineer a particular metabolic phenotype in plants. A key step in lysine synthesis is carried out by dihydrodipicolinate synthase, which is feedback inhibited by the pathway end product, lysine, and thus plays a key role in regulating flux through the pathway. Engineering plants to overexpress a feedback insensitive bacterial dihydrodipicolinate synthase, similar to the approach with the enzyme ADP glucose pyrophosphoralyase (ADPGPP) described below, greatly increased flux through the lysine biosynthetic pathway. However, in most cases this did not result in increased steady-state lysine levels as the plants also responded by increasing flux through the lysine catabolic pathway. Substantial increases in lysine only occurred in plants where flux increased to such a level that the first enzyme of the catabolic pathway became saturated.

Carbohydrate

Plants make both polymeric carbohydrates like starch and fructans, and individual sugars like sucrose. The biosynthesis of these compounds is sufficiently understood to allow the bioengineering of their properties, or to engineer crops to produce polysaccharides not normally present.

Genes responsible for the synthesis of fructans can be used to modify plants of higher agromomic value to produce this polymeric carbohydrate.  Fructans are an important ingredient in functional foods as they promote a healthy colon and help reduce the incidence of colon cancer. The crop of predominant interest for elevated fructan production is the sugar beet, because the major storage compound of this species is sucrose, the direct precursor for fructan biosynthesis. Andries J. Koops, of Wageningen, The Netherlands, has reported high level fructan accumulation in a transgenic sugar beet, achieved by expression of a Jerusalem artichoke gene encoding 1-sucrose:sucrose fructosyl transferase (which mediates the first steps in fructan synthesis) Despite the storage carbohydrate having been altered, there was no visible effect on phenotype and did not affect the growth rate of the taproot as observed under greenhouse conditions (Sévenier, 1998).  Their work has implications both for the commercial manufacture of fructans and also for the use of genetic engineering in obtaining new products from existing crops.

A similar approach is being used to derive soybean varieties that contain some oligofructan components which, by altering the composition of the microflora in the digestive system selectively increase the population of beneficial species of bacteria (e.g., bifidobacteria) in the intestines of humans & certain animals, and out compete the harmful species of bacteria (e.g., E. coli 0157:H7, Salmonella SE, etc.). Thus, high oligofructan soybeans have the potential to displace some of the antibiotics that are historically utilized to combat disease caused by those bacteria.   A collateral effect will be assisting the prevention of the therapeutic overuse of antibiotics, which is the proven source of selection for antibiotic resistance in pathogenic bacteria.    An additional effect could be the creation of environmental conditions preferential to the growth of beneficial strains of bacteria that emit certain short-chain fatty acids.  There are three short-chain fatty acids: acetic acid (2 carbons), propionic acid (3 carbons) and butyric acid (4 carbons). When dietary fiber or other unabsorbed carbohydrates are fermented by colonic bacteria, the products are these short-chain saturated fatty acids. Studies suggest these short-chain fatty acids may enhance absorption of minerals such as iron, calcium, and zinc. Some research has found that propionic acid inhibits cholesterol synthesis in vitro, but this has not been confirmed in human studies (Watkins, 1999).

The soluble oligosaccharides, stachyose and raffinose, can cause flatulence and digestive problems (Hartwig et al., 1997), producing discomfort in humans. For soybean or soybean meal used for livestock feed, the oligosaccharides raffinose and stachyose are not digested by monogastric animals producing a loss of feed efficiency. Mature soybeans from traditional varieties contain 1.4% to 4.1% stachyose. Instead of being digested in the stomach, it passes to the intestines where bacteria ferment it into gases that make people and animals feel unpleasantly full.  In low-stachyose soybeans, stachyose is replaced with the easily-digested sugar sucrose, so low-stachyose soy is also higher in energy content than traditional soy; making it doubly useful as an ingredient in foods, petfoods, and animal feeds.   The increased sucrose content means that low-stachyose soyfoods taste sweeter than their traditional counterparts.

Engineering starch content in potatoes is also of interest. Monsanto have introduced a gene to modify the starch metabolic pathway in potatoes by introducing a new gene which has an endogenous counterpart.  The idea of the new gene is that because it is from another source and under the control of a different promoter its activity is not subject to the same degree of inhibition by the plants native regulatory machinery. This new gene from E. coli codes for the enzyme ADPGPP, and when introduced into potatoes under the control of the tuber-specific patatin promoter, led to a 30% increase in starch content (Stark, 1992). The results of careful consideration of enzyme kinetics in metabolic engineering were elegantly demonstrated in this research directed at modifying starch synthesis by manipulating ADPGPP. Plant ADPGPP is sensitive to allosteric effectors and has been proposed to be a key regulator limiting starch synthesis. Escherichia coli ADPGPP is involved in glycogen synthesis and is also sensitive to allosteric effectors. Mutations affecting allosteric regulation cause an increase in glycogen levels in E. coli. Stark et al. (1992) engineered wild-type and mutant E. coli ADPGPP for expression in plants and assayed the effect on starch accumulation. Tubers from potato plants transformed with the wild-type E. coli enzyme had starch levels similar to wild-type plants, whereas those transformed with the allosterically insensitive E. coli ADPGPP enzyme had starch levels up to 60% higher than wild type. This has an added bonus as the higher starch content results in a lower moisture content leading to far less fat absorption on frying, as moisture lost during frying is replaced by oil uptake. The effect was only observed when the mutant protein was targeted to the chloroplast and driven by a tuber specific promoter; constitutive expression was lethal. Such results demonstrate the importance of considering the target tissues, subcellular localization, and kinetics of enzymes when engineering plant metabolism.. 

Starch is used in a wide range of industrial applications such as coatings for paper and textiles, as a gelling agent in the food industry.  It is now possible to make some high value starches for example starches that are free of the amylose fraction making the generic product more valuable. In the past two years, large amounts of genetically modified potatoes producing an amylose-free starch have been produced.  This will be the first example of genetically engineered starch with superior quality over traditional starches entering the markets. It is likely that starches with other alterations will follow in the next few years.  Examples will be starches with an altered amylopectin chain length distribution or a modified phosphate content, as it is possible now to specifically engineer these traits.  It can also be envisioned that a broad range of novel starches will be produced through combining the down regulation or over expression of several genes.

Fiber

Fiber, or roughage, is a group of substances chemically similar to carbohydrates. It is only found in foods derived from plants, and never occurs in animal products. Fiber provides bulk in the diet, so foods rich in fiber fill you up without contributing excessive calories. Current controversies aside, there is ample scientific evidence to show that prolonged intake of dietary fiber has positive health benefits, especially, inter alia, for reduced risk from colon and other types cancer. How the intake of fiber works remains a mystery. Research is needed on the mode of action and on the differential benefits of various forms of fiber.

Fiber type and quantity are undoubtedly under genetic control, although this topic has been little studied. The technology to genetically manipulate fiber content and type would be a great benefit to the health status of many individuals who refuse, for taste or other reasons, to carefully include fiber in their daily diet.  For example fiber content could be added to more preferred foods, and thus facilitate dietary fiber intake. The more common sources of dietary fiber could be altered for greater health benefits.

Schwall et al (2000) has created by genetic engineering a potato producing very high amylose (slowly digested) starch by inhibiting two enzymes that would normally make the     amylopectin type of starch that is rapidly digested. The range of starch applications is heavily influenced by the ratio of its two major components, essentially linear amylose and branched amylopectin, the length and distribution of the branch chains and phosphate levels, and nonstarch components like lipids and proteins. The ratio of amylose to amylopectin has the greatest influence on the physicochemical properties of the starch, and for many applications it is desirable to have a pure or enriched fraction of either amylopectin or amylose. In most crops starch contains 20–30% amylose and 70–80% amylopectin. The antisense simultaneous inhibition of a minor form of starch branching enzymes, SBE A and B results in very-high-amylose potato starch containing insignificant levels of highly branched amylopectin.

Fermentation of fiber to release volatile fatty acids, including acetic, propionic and butyric                     acids, has been recognized for some time to be beneficial to the health and absorptive functioning of the intestinal cells lining the lower bowel. Work by German at UC Davis (Watkins, 1999) has shown the fermentation of fiber to be associated with the beneficial effects of fiber towards lower bowel cancer. Parallel studies on the mechanisms of action of short chain fatty acids toward cancerous cells has shown that butyric acid has an unusual activity in modifying the structure of nuclear proteins that apparently causes cancerous cells to die by the process of apoptosis. 

 Micro Nutrients

Secondary Plant metabolites – Nutraceuticals

  The relentless search for new compounds to treat human disease has led to the formation of specialized biotechnology firms searching for nutraceuticals, ie foods or parts of foods that are believed to have medicinal value. With micronutrients, when we talk about vitamins and minerals in our diet we can think about two levels: the Recommended Daily Allowance or RDA and levels in excess of the RDA that are associated with additional beneficial or therapeutic effects. RDAs are the minimum recommended intake needed to alleviate nutrient deficiency, and are somewhat misleading, as they are not the levels needed for optimal health. Indeed, RDAs do not reflect the growing knowledge base indicating that the elevated intake of specific vitamins and minerals (for example, vitamins E and C, carotenoids, and selenium) significantly reduces the risk of diseases such as certain cancers, cardiovascular diseases, and chronic degenerative diseases associated with aging. In order to obtain such therapeutic levels in the diet, additional fortification of the food supply will be required as well as modification of dietary preferences, or direct modification of micronutrient levels in food crops.

 For select mineral targets (iron, calcium, selenium, and iodine) and a limited number of vitamin targets (folate, vitamins E, B6, and A), the clinical and epidemiological evidence is clear that they play a significant role in maintenance of optimal health and are limiting in diets worldwide. Unlike vitamins and minerals, the primary evidence for the health-promoting roles of phytochemicals comes from epidemiological studies, and the exact chemical identity of many active compounds has yet to be determined. However, for select groups of phytochemicals, such as non-provitamin A carotenoids, glucosinolates, and phytoestrogens, the active compound or compounds have been identified and rigorously studied. A great irony of nature is that the body's natural metabolism involving oxygen also produces a host of toxic compounds called "free radicals." These compounds can harm body cells by altering molecules of protein and fat, and by damaging DNA, the cell's genetic material. Antioxidants counteract, or neutralize, the harmful effects of free radicals.

Vitamin E levels are being improved in several crops, including soybean, maize and canola, while rice varieties are being developed with the enhanced Vitamin A pre-cursor beta-carotene for which deficiency has tragic consequences in many developing countries. Other targets include improved iron content, through the production of iron-rich storage protein, and phosphorus in the form of phytate, and isoflavonoids.

 As with macronutrients, one way to ensure an adequate dietary intake of nutritionally beneficial phytocompounds is to manipulate their levels in plant foods.  Until recently such work had been hindered by the difficulty in isolating the relevant genes (eg. for vitamin biosynthesis). However, the advent of genomics during the past 5 years has provided new routes for such work.  One aspect of genomics is the complete sequencing of an organism's entire genome.  This means that genes for vitamin synthesis from simple organisms like bacteria and fungi can be used to rapidly identify vitamin biosynthetic genes in more complex organisms like plants.  In the past several years, Dean Della Penna’s laboratory (Michigan State University) have developed and applied this approach called Nutritional Genomics, to dissect and manipulate the synthesis of Vitamin E in plants. Vitamin E is the most important fat-soluble antioxidant in our diet; cannot be synthesized by humans and must be obtained from plant sources in our diet. Unfortunately, obtaining the required amount from the average diet is extremely difficult.  The reason for this is that the major vitamin E sources in our diets, plant oils, contain vitamin E precursors that are 10 to 50 times less active than the most active form of the vitamin, alpha-tocopherol.  Indeed, soy and maize oils contain 90% and 60%, respectively, of their potential vitamin E as these low activity precursors. Using Nutritional Genomics Della Penna (1999) isolated a gene (gamma-tocopherol methyltransferase (g-TMT) that can convert the lower activity precursors to the highest activity Vitamin E compound, alpha-tocopherol.  With this technology they have increased the vitamin E content of Arabidopsis seed oil nearly 10-fold and are now working with industry to move the technology to agricultural crops such as soybean, maize and canola. Engineering similar conversions in soybean, canola and maize would elevate the levels of this important antioxidant/vitamin in the diet and potentially have significant health consequences for the general population.

 Vitamin A is a highly essential micronutrient and widespread dietary deficiency of this vitamin in rice-eating Asian countries has tragic undertones: five million children in South East Asia develop an eye disease called xerophthalmia every year, and 250,000 of them eventually become blind.  Improved vitamin A nutrition would alleviate this serious health problem and, according to UNICEF, could also prevent up to two million infant deaths because vitamin A deficiency predisposes them to diarrhea diseases and measles. Flowers and fruits owe their dazzling colors to carotenoid pigments. Beta-carotene, the best-known carotenoid, which gives carrots and sweet potatoes their orange color, is a precursor to vitamin A. Though the carotenoid biosynthetic pathway in plants had been known since the mid 1960s, the labile, membrane-associated enzymes remained recalcitrant to isolation and study. However, because carotenoids are also synthesized by many photosynthetic and non-photosynthetic bacteria, the development of molecular genetic tools in prokaryotes during the 1980s allowed plant researchers to access carotenoid biosynthetic genes from prokaryotes. Integrating prokaryotic systems into their work enabled researchers to finally clone the majority of carotenoid biosynthetic enzymes from plant during the 1990s.

 Rice is a staple that feeds nearly half the world's population, but milled rice does not contain any beta-carotene or its carotenoid precursors. A research team led by Peter Burkhardt and Ingo Potrykus of the Swiss Federal Institute of Technology in Zurich, in collaboration with scientists from the University of Freiburg in Germany, discovered, however, that immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate of beta carotene biosynthesis (Ye, 2000).  Condensation of two such molecules produces a 40-carbon molecule called phytoene, the first carotenoid precursor in the biosynthetic pathway leading to the production of beta-carotene. Potrykus not only stitched on the next series of enzymatic steps but ensured that they were directed to the correct site of synthesis where the precursor (geranylgeranyl-diphosphate) is formed in the endosperm plastids. He achieved this by cleverly including a functional transit peptide from an enzyme (Rubisco) used in photosynthesis thus allowing plastid import.  The genes were in the following order: Phytoene synthase (from Daffodil), - Phytoene desaturase (Erwinia uredovora ),  - Lycopene beta-cyclase (from Daffodil). Two of the genes were from daffodil (narcissus) the middle one was from a bacteria - the reason being that in one step it completes a function that requires three additional steps in the daffodil - scientists will always try to work with the system that makes most sense and thus are often the subject of condemnation for their resourcefulness.  This major breakthrough shows that an important step in provitamin A synthesis can be engineered in a non-green plant part that normally does not contain carotenoid pigments. A similar method was used by Monsanto to produce beta-carotene in canola. 

 Taking a different tack, at the Hebrew University in Jerusalem, Joseph Hirschberg has induced tobacco plants to make a carotenoid pigment called astaxanthin, which can be used to tint flowers, farm-raised shrimp, and salmon and, when fed to chickens, can color egg yolks a vibrant orange. Currently, astaxanthin is extracted from seashells or synthesized chemically and carries a price tag of about $2600 per kilogram. But Hirschberg's efforts may help bring that price down.  Other interest in the carotenoid pathway products include lycopene which has suggested cardio vascular and prostate effects, Lutein/Zeaxanthin which have suggested effects on the prevention of macular degeneration and of course vibrant yellow/orange red colorants for everything from margarine to flowers!

 Iron is the most commonly deficient micronutrient in the human diet and iron deficiency affects an estimated 1-2 billion people.  Anemia characterized by low haemaglobin is the most widely recognized symptom of iron deficiency, but there are other serious problems such as impaired learning ability in children, increased susceptibility to infection and reduced work capacity.  Women of childbearing age are especially prone to iron deficiency and suffer from tragic consequences such as premature child birth, babies with low birth weight and even greater risk of death.

 Increasing the iron content in rice is an appealing strategy to supply the mineral inexpensively and effortlessly to a large sector of the world's disadvantaged population.  Rice feeds half of the world, and is eaten every day in those parts of the world where iron deficiency is most prevalent. A research group led by Toshihiro Yoshihara and Fumiyuki Goto at the Central Research Institute of Electric Power Industry in Japan employed the gene for ferritin, an iron-rich soybean storage protein, under the control of an endosperm-specific promoter.  Grains from transgenic rice plants contained three times more iron than normal rice. Potrykus’ team in Zurich, has developed similar transgenic rice with the ferritin gene from beans, and the plants are now being evaluated. The cultivar transformed for higher iron, containing ferritin, an iron-rich bean storage protein, a fungal phytase, an enzyme that breaks down phytate making Fe available, and to prevent reabsorption of iron, a gene for a cystein-rich metallothionein-like protein has yet to be crossed with the beta- carotene one.  To further increase the iron content in the grain they plan to focus on iron transport within the plant (Potrykus, I. (1999)

 Isoflavones have drawn much attention because of their benefits to human health.  The isoflavones genistein and daidzein are naturally occurring plant compounds that are being studied for their substantial health benefits. They are found almost exclusively in soybeans and other leguminous plants. The reported health benefits include exhibiting estrogenic and anticancer activity, help prevent artherogenic oxidation of low density lipoproteins, and have positive effects on improving bone mass. The basis for these effects has not been established, but the weak estrogenic activity of isoflavones, which are sometimes referred to as phytoestrogens, may be a factor in conferring these properties. As a result, many food manufacturers are striving to provide products containing soy and/or isoflavones to consumers. Soybean seeds and protein products produced from seeds are the primary source of isoflavones in the human diet. The isoflavone content in soybean seeds varies depending on the variety and environmental conditions when grown. Losses of isoflavones due to processing of seeds for traditional soy foods or protein products can reach 50% or more. Together these factors can contribute to difficulties in reaching efficacious levels of isoflavones in soy products. This problem could be addressed by increasing isoflavone concentrations and reducing their variability in soybean seeds.

 These isolflavones, which are produced almost exclusively in legumes, have natural roles in plant defense and root nodulation. Isoflavone synthase catalyzes the first committed step of isoflavone biosynthesis, a branch of the phenylpropanoid pathway. To identify the gene encoding this enzyme, a group (REF) used a yeast expression assay to screen soybean ESTs encoding cytochrome P450 proteins. They identified two soybean genes encoding isoflavone synthase, and used them to isolate homologous genes from other leguminous species including red clover, white clover, hairy vetch, mung bean, alfalfa, lentil, snow pea, and lupine, as well as from the non-leguminous sugar beet. They expressed soybean isoflavone synthase in Arabidopsis thaliana, which led to production of the isoflavone genistein in this nonlegume plant. Identification of the isoflavone synthase gene should allow manipulation of the phenylpropanoid pathway for agronomic and nutritional purposes..  Of the enzymes necessary for engineering isoflavone nutraceuticals into plants, only two, the 2-hydroxylase and dehydratase of the isoflavone synthase complex, have yet to be characterized at the molecular level.  The dimeric lignans similarly have potent anticancer and antioxidant activity, and genes encoding all the enzymes for the conversion of coniferyl alcohol to secoisolariciresinol, a major dietary phytoestrogen, have been cloned.  These include the remarkable dirigent protein that co-acts with oxidases to confer stereochemical free radical coupling, and the (+)-pinoresinol/(+)-lariciresinol reductase that shares extensive sequence similarity to legume isoflavone reductases. Expected in 2005 are soybeans derived via biotechnology to contain elevated four time amounts of isoflavones. 

 From a counter perspective, isoflavones have been shown to impart a negative taste component to foods and the reduction of isoflavone concentrations would be of value for other products.  Another benefit of manipulating isoflavone synthase expression in legume and nonlegume crop species is that increased levels of isoflavones may increase resistance to various pathogens. Developing other grain crops that can synthesize isoflavones would provide food manufacturers with alternatives to soy for use in their products.

 Research, funded by the Kansas Wheat Commission in the laboratory of Dolores Takemoto, a biochemist at   Kansas State University in Manhattan report that some kinds of wheat are capable of inhibiting the growth of colon cancer cells in laboratory tests, as well as in trials using mice. The scientists compared 150 different types of wheat and found those high in the antioxidants caffeic acid and ferulic acid were the most potent cancer cell fighters. The wheat gene responsible for producing these antioxidants is   known and it will be possible to genetically engineer wheat to be rich in caffeic and ferulic acids, (health and Wellness).

 Utilizing chalcone synthase and dihydroflavonol reductase constructs, it has been possible to alter the content and composition of condensed tannins in birdsfoot trefoil clover (Lotus corniculatus).  These studies are important because condensed tannins are believed to help prevent bloat in ruminants feeding on highly digestible forages.  More global upregulation of phenylpropanoid biosynthesis by over-expression of L-phenylalanine ammonia-lyase results in increased local and systemic resistance of tobacco to microbial pathogens, but compromised systemic resistance to herbivorous insect larvae.  This underlines the potential for unexpected metabolic cross-talk during genetic manipulation of natural product pathways.

 Anti-Nutrients

Seeds store the phosphorous needed for germination in the form of phytate, a sugar alcohol molecule having six phosphate groups attached.  In terms of food and feed, though, phytate is an anti-nutrient because it strongly chelates iron, calcium, zinc and other divalent mineral ions, making them unavailable for uptake.  Potrykus and his group have developed a series of transgenic rice lines designed to deal with this problem.  One approach has been to reduce the phytate in rice endosperm by introducing a gene from the fungus Aspergillus niger that encodes phytase, an enzyme that breaks down phytate. To counter phytate from other sources in the diet, the Swiss group is using another gene that encodes for a heat-stable phytase from Aspergillus fumigatus.  This enzyme can survive boiling and has two pH optima - acidic for the stomach and alkaline for the intestine.  To further promote the reabsorption of iron, a gene for a metallothionein-like protein has also been engineered. Potrykus commented that all these transgenics will soon be tested and eventually the traits will be combined into a multiply-engineered line.

 Phytate also has implications for animal nutrition. A team of scientists at the University of Wisconsin and the USDA-ARS Dairy Forage Research Center (Madison, Wisconsin) has genetically engineered alfalfa to produce phytase.  The resulting transgenic alfalfa lines performed well when grown in the field, with no yield reduction.  In a poultry feeding trial, better results were obtained using transgenic plant material than with the commercially produced phytase supplement.  Poultry grew well on the engineered alfalfa diet without any inorganic phosphorus supplement, which shows that plants can be tailored to increase the bioavailability of this essential mineral.  Thus phosphorus supplements can be eliminated from poultry feed, which could reduce costs and mitigate the problem of phosphorus pollution. Children require phosphate in their diet for proper growth.  However, the phosphate naturally present in traditional varieties of soybeans and corn exists primarily in the form of an insoluble phytate (chemically bound as phytic acid). 

 Monogastric animals such as humans (also poultry and swine) lack the phytase enzyme needed for digestion of phytate (also known as inositol hexaphosphate).  Thus most of the extant corn and soy phytate is excreted by humans/animals, which can sometimes cause water pollution problems.  Poultry and swine producers in most countries currently add mined & processed (powdered) phosphate to their feed rations to enable optimal growth of those animals.  When low-phytate soybean meal is utilized along with low-phytate maize to manufacture animal feed rations, the phosphate emissions in swine and poultry manure are reduced by halfLow-phytate maize was commercialized in the US in 1999.  Low-phytate soybeans have already been created, but the seed companies are still working to achieve an acceptable yield per hectare, before these soybeans are commercialized. Research indicates that the protein in low-phytate soybeans is also slightly more digestible than the protein in traditional soybeans. Phytase supplementation improved Ca and P digestibilities to varying degrees. Supplementation of phytase in normal, corn-soybean meal diets improved feed intake, feed conversion, and egg mass and elicited a response in shell quality and egg components at the low (0.10%) Non-Polysaccharide Proteins. .

 In response to the critical need of the swine industry to reduce manure-based enviroment pollution Serguei Golovan (Golovan, S, 2001) produced Wayne (Enviropig) the low-phosphate pig who has a transgenic phytase gene (PSP/APPA transgene) that specifies the secretion of phytase enzyme in the saliva that digests phosphorus from phytate, the most abundant source of phosphorus in hog diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important pollutant of hog production, and ironically, mineral phosphate must be added to the ration to meet the nutritional requirement for phosphorus. Animals from all of these lines exhibit significantly enhanced digestion of phytate phosphorus and reduced fecal phosphorus output by up to 75%. These are the first transgenic animals designed to meet an environmental objective and the first transgenic pigs produced in Canada. They have the potential to make a significant impact on the management of phosphorus nutrition and environmental pollution in the hog industry.

 As noted, phytate also tends to chemically “bind” some of the iron, calcium, and zinc in prepared food products, thus making a portion of those minerals present in foods unavailable to be digested.  That is why U.S. regulations mandate that a 20% nutritional excess of these minerals be added to infant formula products. In addition to reducing the phosphate pollution load on sewage processing facilities, widespread use of low-phytate soy and maize and corn in food products would reduce the need for additional small amounts of minerals to be added to some processed food products for children.

  For those (adults) who want to minimize their risk of developing cancer of the colon, breast, prostate or liver, the consumption of large amounts (e.g., 2 to 4 grams per day) of phytate has been shown to apparently inhibit initiation of those cancers. Because it is difficult to consume that level of phytate in foods made from traditional soybean varieties, high-phytate soybeans developed via biotechnology would be one way to more easily incorporate that apparent cancer preventative in the adult human diet. 

  Allergens

  While symptoms of food intolerance are common, true food allergy is less common. A food allergy is distinguished from food intolerance and other disorders by the production of antibodies (IgEs) and the release of histamine and similar substances. The immune system produces antibodies and substances including histamine in response to ingestion of a particular food or food component. The symptoms may be localized to the stomach and intestines, or may involve many parts of the body after the food is digested or absorbed. The symptoms usually begin immediately, seldom more than 2 hours after eating. The best characterized true allergens include the superfamily cupins which include globulins found in nuts and beans, albumins in nuts; the superfamily prolamins found in cereals and others such as heverin which causes contact dermatitis from latex and chiinases.

  Foods that more frequently cause malabsorption or other food intolerance syndromes other than direct immune responses include: wheat and other gluten-containing grains (celiac disease), cow’s milk (milk/lactose intolerance and intolerance of dairy products) corn products.  Work by Bob Buchannan in Berkeley has indicated that extensions of the biochemical and molecular studies have led to the potential application of the gene thioredoxin in reducing allergencity.  According to present evidence, thioredoxin may be used to improve foods through, among other changes, lowering allergenicity and increasing digestibility. Reduction by thioredoxin changes biochemical and physical properties of proteins. Using a canine model system, Buchannan’s laboratory has shown that thioredoxin reduces and thereby alters the allergenic properties of disulfide proteins extracted from wheat flour.   By changing the levels of expression of the thioredoxin gene scientists have been able to reduce the allergenic effects of wheat and other cereals. Alternatively, if the allergen is know scientists can engineer plants that have their allergen genes turned off.

  The list of anti-nutritional components found in soybean meal and soyfoods includes the trypsin inhibitors, lectins, and several heat-stable components. The trypsin inhibitors in soybeans(Bowman, 1944; Kunitz, 1945) are the major anti-nutritional components of this protein source. Since the trypsin inhibitor is a sulphur rich protein, null or low lines will be slightly deficient in essential amino acids, particularly the sulphur-rich amino acids which can be countered by introducing the Mb1 protein ( see above). One protein (p34) accounts for 85% of anti-soy IgEs in soy-sensitive individuals.  Sense co-suppression inhibits the accumulation of p34 in transgenic soybean seeds removing the principal source of food allergenicity  in soy (E. Herman, USDA; T. Kinney, Dupont).Reduced allergens, trypsin inhibitors and increased limiting amino acids levels in soybeans would have a positive impact on the domestic feed industry and offer a competitive advantage for on-farm feeding of this protein source.

  Toxins:

  Plants are not benign and produce many interesting phytochemicals to protect themselves from marauding pests. Over years of breeding and selection most of the noxious genes have been weeded out. Potatoes and tomatoes are members of the deadly nightshade families and contain toxic glycoalkaloids, which have been linked to spina bifida; kidney beans contain phytohaemagglutinin and are poisonous if undercooked.  Dozens of people die each year from cynaogenic glycosides from peach seeds and grayanotoxin in honey produced from the nectar of rhododendrons.  Biotechnology approaches can be used to downregulate or even eliminate the genes involved in the metabolic pathways for the production of these toxins in plants.

 Challenges

  Metabolic engineering to plants is hindered by a lack of quantitative information on fluxes in the metabolic pathways. Analysis of fluxes in metabolic pathways in response to an environmental or genetic manipulation can help identify rate-limiting steps. Since total productivity is the goal for enhanced production of a valuable product, the product of the biomass productivity times the metabolite specific yield should be optimized. This implies a systems analysis of both primary and secondary metabolic pathways. The manipulation of what are considered to be well-characterized "rate-limiting" enzymes of primary carbon metabolism to study their role in regulating pathway flux has provided some of the more surprising results from metabolic engineering in plants. These experiments drive home the point that a thorough understanding of the individual kinetic properties of enzymes may not be informative as to their role in complex metabolic pathways. Potential regulatory enzymes are generally identified based on their catalyzing irreversible reactions and being regulated by appropriate effector molecules for a pathway; traditional biochemical hallmarks of rate controlling enzymes. When the highly regulated Calvin cycle enzymes Fru-1, 6-bisphosphatase and phoshphoribulokinase were reduced 3- and 10-fold in activity, respectively, surprisingly minor effects were observed on the photosynthetic rate. In contrast, a minor degree of inhibition of plastid aldolase, which catalyzes a reversible reaction and is not subject to allosteric regulation, led to significant decreases in photosynthetic rate and carbon partitioning. Thus aldolase, an enzyme seemingly irrelevant in regulating pathway flux, was shown to have a major control over the pathway. Analogous surprises were also found when manipulating presumed "rate limiting" enzymes of glycolysis. Such data has called into question many of the longstanding ideas about flux regulation in plants and is forcing a reassessment of the role of individual enzymes in the process. These studies also make clear the caution that must be exercised when extrapolating individual enzyme kinetics to the control of pathway flux.

 In addition consideration needs to given to the site of synthesis and site of activity of the enzyme as Potrykus did with the beta-carotene pathway. However signal sequences or transit peptides are not always sufficient to insure targeting, for example in plastid transportation charge and size may also matter.  Another problem that is a potential challenge in biological systems is redundancy of pathways. For example,  4-Coumarate:CoA ligases (4CLs) are a group of enzymes necessary for maintaining a continuous metabolic flux for the biosynthesis of plant phenylpropanoids, such as lignin and flavonoids, that are essential to the survival of plants. So far, various biochemical and molecular studies of plant 4CLs seem to suggest that 4CL isoforms in plants are functionally indistinguishable in mediating the biosynthesis of these phenolics. However,  Hu et al (1999) showed that the expression of the isoform Pt4CL1 and Pt4CL2 genes is compartmentalized to regulate the differential formation of phenylpropanoids that confer different physiological functions in aspen. When Vincent Chiang used an antisense construct to turn off the 4CL a pivotal gene in the lignin pathway in aspen (lower lignin = cheaper paper and less waste) he demonstrated a profound effect on wood composition and tree growth: At 10 months of age, the transgenic aspens contained up to 45% less lignin and as much as 15% more cellulose than nontransgenic aspens. However, the lignin content, not composition, was altered. The fact that the extent of growth enhancement was not directly correlated with lignin content prompted the authors to propose that other pathways other than lignin biosynthesis may be involved.

 An intriguing approach for metabolic engineering and increasing our understanding of the coordinate changes in gene expression needed to regulate entire pathways is to identify and study transcriptional factors controlling pathways or branches of metabolism. Many of the transcriptional regulators affecting plant biochemistry and development were originally identified by chemical- or transposon-based mutant screens in maize, snapdragon or Arabidopsis. The cloning of such loci has provided the opportunity to use these genes to manipulate plant biochemistry in the host organism or in other plants. One of the early instances of using this approach to manipulate plant biochemistry was the engineering of Arabidopsis to express the maize transcription factors C1 and R, which regulate production of anthocyanins in maize aleurone layers. Expression of C1 and R together from a strong promoter caused massive accumulation of anthocyanins in Arabidopsis, presumably by activating the entire pathway. More recently, the maize transcriptional regulators C1, R, and P were expressed in cell cultures and the effect on anthocyanin biochemistry and global gene expression analyzed. Novel insights into the anthocyanin pathway, its regulation, and additional differentially expressed targets of these regulatory genes were obtained. Such expression experiments hold great promise and may eventually allow the determination of transcriptional regulatory networks for biochemical pathways.

 Research to improve the nutritional quality of plants has historically been limited by a lack of basic knowledge of plant metabolism and the almost insurmountable challenge of resolving complex branches of thousands of metabolic pathways.  With the tools now available to us through the field of genomics and bioinformatics, we have the potential to fish “in silico” for genes of value across species, phyla and kingdoms. And subsequently to  study the expression and interaction of transgenes on tens of thousands of endogenous genes simultaneously.  With advances in proteomics we should also be able to simultaneously quantify the levels of many individual proteins or follow post-translational alterations that occur. Although metabolomics has been coined to describe the study of the complex circuitry beyond the proteomics level – at this point that is all it is – a name.  The paper by Gavin et al discussed at the beginning is very promising in that it demonstrates that the tools are now being developed to allow us to analyze interactions at this crucial level. With these newly evolving tools we are beginning to get a handle on global effects of metabolic engineering on metabolites, enzyme activities and fluxes.  When this becomes possible the increase in our basic knowledge of plant secondary metabolism during the coming decades will be truly unparalleled and will place plant researchers in the position of being able to modify the nutritional content of major crops to improve aspects of human and animal health.  For essential minerals and vitamins that are limiting in world diets, the need and way forward is clear, and improvement strategies should be pursued, as long as attention is paid to the upper safe limit of intake for each nutrient.  However, for many other health-promoting phytochemicals, decisions will need to be made regarding the precise compound or compounds to target and which crops to modify such that the greatest nutritional impact and health benefits are achieved.  Because these decisions will require an understanding of plant biochemistry, human physiology, and food chemistry, strong interdisciplinary collaborations will be needed among plant scientists, human nutritionists, and food scientists in order to ensure a safe and healthful food supply for this new century ( della Penna, 1999).