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Modern Ag Biotech Applications Martina Newell-McGloughlin

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1 Modern Ag Biotech Applications Martina Newell-McGloughlin
Original file of this presentation Martina Newell-McGloughlin Director, UC Systemwide Biotech Research and Education Program

2 Crop Biotechnology Agronomic Traits Biotic Stress
Insect Resistance – Bt, cystatin Disease Resistance Viral- coat protein protection (Papaya ringspot virus) Bacterial, Fungal, Nematode (Rice blight, rice blast) Weed- herbicide tolerance (Striga, orobanchia) ABiotic Stress Drought, Cold, Heat Poor soil Salinity – tomatoes with transport protein Aluminum -Citric acid Yield Nitrogen Assimilation – nodulation by rhizobia, GDH metab eng Starch Biosynthesis, O2 Assimilation, photosynthesis/Rubisco Quality Traits Processing Post harvest loss reduction Reproduction: sex barriers, male sterility, seedless fruit Nutrients (Nutraceuticals) Macro: Protein (Cassava-ASP), Carbohydrates, Fats, Fiber Micro: vitamins, minerals, phytochemicals Anti-nutrients: Phytase, Toxin removal Novel Crop Products Proteins: nutraceuticals, therapeutics, vaccines Renewable resources: Biomass conversion, feedstocks, biofuels, phytoremediation PROGRESS TO DATE Genetic modifications of crop plants can be organized into two broad-based non-mutually exclusive categories: those that benefit the producer and those that benefit the consumer. Modifications that protect the crop from either biotic stress (e.g., damage by predators such as insects and nematodes; diseases caused by viruses, fungi, bacteria; and weeds) or abiotic stress (e.g., drought, cold, heat, and poor soils) or increase total crop yield benefit the producer and are called “input traits.” The majority of modified crops in commercial use fit in this group. Scientists have just begun to tap the large potential of biotechnology to produce varieties of plants that confer a wide spectrum of advantages to consumers. These varieties are modified with “output traits.” The estimated global area of transgenic or genetically modified (GM) crops for 2001 is 52.6M ha (130.0 million acres), grown by 5.5 million farmers in thirteen countries. In 2001, four principal countries grew 99% of the global GM crops.. The USA grew 35.7 million ha (68% of global total), followed by Argentina with 11.8 million ha (22%), Canada with 3.2 million ha (6%) and China with 1.5 million ha (3%); China had the highest year-on-year percentage growth with a tripling of its Bt cotton area from 0.5 million ha in 2000 to 1.5 million ha. in 2001 (other crops acreage is ). Globally, the principal GM crops were soybean ( 33.3 million ha, 63% of global area, maize (9.8 million ha 19%), cotton (6.8 million ha 13%), and canola (2.7 million ha 5%). The breakdown by country, crop and trait is illustrated in Figure 1 & 2 and Table 1 and 2. ( James, 2001) In the first 6 years since introduction, 1996 to 2001, a cumulative total of over 175 million ha (almost 440 million acres) of GM crops were planted globally and met the expectations of millions of large and small farmers. Rapid adoption and planting of transgenic crops by millions of farmers around the world, growing global political, institutional, and country support for biotech crops, and data from independent sources confirm and support the benefits associated with biotech crops (James, 2001). During this early phase of the plant revolution, the benefits of plant genetic engineering have been largely confined to farmers. From a consumer position however here has been a major reduction of up to 93% in the mycotoxin fumonosin on Bt maize as reduction in insect damage limits access to fungal spores. Currently, U.S. companies that are active in transgenic plant research spend far more money on research and development than they will receive as their share of profits from modified seeds, sales of which will amount to approximately $1.5 billion. Most companies, including those in Europe, envision a far more lucrative future when the plant revolution matures further. One possibility is the $500 billion market for foods in the United States. The next major phase of the plant revolution is emphasizing the engineering of value added traits in plants. These traits will be ones that are readily apparent to the consumer. Adoption of the next stage of biotech crops may proceed more slowly, as the market confronts issues of how to determine price, share the value, and adjust marketing and handling to accommodate specialized end-use characteristics. Furthermore, competition from existing alternative products will not evaporate. Pitfalls that have accompanied the first generation of biotech crops, such as the US trade dispute with Europe and Japan over approval and labeling of GM crops, will also affect the next stage of products. Some industry analysts believe the development of more end-use quality traits will largely dismantle the existing marketing system of “commodity” field crops. In other words, there will be a movement away from bulk handling and blending of undifferentiated crops under very broad grades and standards categories and toward a system that can meet more specialized needs of buyers, even to the point of preserving the identity of a crop from the farm to the user. The added costs of such specialized handling will have to be justified by the value of the new crops to buyers. The leading commodity in this value-added focus area is the field broadly defined as “functional foods.” The Institute of Medicine’s Food and Nutrition Board (IOM/NAS, 1994) defined functional foods as “any food or food ingredient that may provide a health benefit beyond the traditional nutrients it contains.” (Goldberg I, 1994 Hasler, 1998). The term nutraceutical was coined by the Foundation for Innovation in Medicine in 1991 (DeFelice, 1991) and is defined as "any substance that may be considered a food or part of a food and provides medical or health benefits, including the prevention and treatment of disease.” The nutraceutical industry has been viewed with renewed interest since the passage in October 1994 of the Dietary Supplement Health and Education Act (DSHEA). Before the passage of the DSHEA, regulations restricted the marketing of nutraceuticals, and companies’ limited their investment in research and development. Modifications in the regulations now make this market more appealing. One estimate states that foods that are used for nutraceutical purposes made up 10% of the value $503 billion total value of the U.S.Retail food market. (Food Labeling News. 1994) One of the major changes brought about by the DSHEA was the change in classification of dietary supplements from "food additives" which require FDA approval to "food" which does not. Another significant change was that dietary supplements might now be labeled with truthful claims about their medicinal benefits. Interestingly, while food supplement manufacturers can label their products with statements about health benefits, food manufacturers who sell foods fortified with the same nutrients may not. In addition, food supplements can now be marketed as conventional foods, which was prohibited under the former regulations.

3 90% resource-poor LDC farmers (12.3 M - 11.0 M 2007) most Bt cotton
Biotech Crops 2008: 125 million hectares (310 million acres up 9.4%) X74 – 25 countries (15 LDC) 12% increase over 2007, 13.3 M farmers (12 M 2007) 90% resource-poor LDC farmers (12.3 M M 2007) most Bt cotton New: Egypt, Burkina Faso, Paraguay , Uruguay. India Bt cotton up to 7.4 M Ha. Source: ISAAA 3

4 Snap shot $44 billion 1996 to 2007, 44% yield gains, 56% reduction costs (including a 359,000 tonne a.i. in pesticides); gains of 141 million tons, would have required 43 million additional hectares Environmental pesticide footprint down by 15.4 %. GM reduction in 286 million kg of CO2 emissions equivalent to removing 6 M cars from the roads (Brookes 2008) HT- increase in no- till: reduction in erosion, soils much healthier, organic matter, less soil compaction, fuel use down by 20 gals/acre (Fawcett & Towery 2005 ) CP papaya saved Hawaii papaya industry (and helped organic farmers!) may be the outcome for plum pox –C5 PTGS insurance against typhoid Mary in nurseries

5 Farmers Choice Papaya ringspot virus
No natural resistance anywhere so could never breed resistance Removes viral reservoir thus protects all growers Field trial of transgenic 'UH Rainbow' and 'UH SunUp' was established in Puna in Gonsalves, Mansardt, Ferreira Plums highly resistant to PPV - System is totally resistant as virus is not harbored unknowingly – Tolerant non biotech trees can harbor virus

6 Snapshot China BT rice GM used pesticides fewer than 1/season; conventional rice used pesticides 3.7 times ( Rozell, 2005) Pesticides cost applied to the conventional rice was 8 to 10 times as high as GM. 80% reduction in pesticide use. Significant decrease in adverse health effects – Lives saved India Bt cotton decrease insecticide use 70% and increased productivity in 58% (737 kg/Ha) Organisms in “Bt crops” fields fared better in trials than those with insecticides Monarch butterflies increase Engineering Modified Bt Toxins to Counter Insect Resistance - pink bollworms cadherin mutation bypass with modifed Cry protein Science, Nov. 2007 BT corn 90% reduction in mycotoxin fungal fumonisins - total US benefit estimated at $23m annually. (Wu, 2006) Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-LRR) -UI study concluded for the major potato-producing regions of the world would be $4.3 billion. 6

7 The Argentinean Case: GM crops an engine of economical development
Number 2 in GM production. 17% of the global area of GM plants. In 2007, 98% of soybean in Argentina was GM. Yield have reached over 6 tonns per hectare In production costs were 182 dolars/Ha; in 2007 are 117 dolars/Ha In farmers spent 78 dolars/Ha in herbicides; today they spend 37 dolars/Ha and insecticide use has decrease 90%. Economical benefits of GM soybean USD$ 20 billion + 1 million jobs Problems, yes. Due to the economic success of GM soybean and maize, President Cristina Fernádez de Kirchne created a new tax on Gm soybean exports that producers oppose 7

8 China is awakening Between the chinese government spent 15 billion US dólars on AgBiotech projects; a 400% increase has been projected National Biotechnology program work on the development of over 130 varieties of GM rice and 55 varieties of GM cotton 10 GM products have been aproved for human consumption (rice, maize, soybean and potato). Bt and disease resistent rice is commercially planted in China. GM cotton was used by 7.1 million small farmers in 3.8 million hectares in 2007 with an economical benefit of USD$ 817 million in 2006. 8

9 Bt corn farmers earn about $85 more per acre, ISAAA (2006), while at the same time producing a healthier feed that is better for the environment. Spain: farmer Jose Victor Nogues” After 5 years of GM crops in the area, most people can appreciate the huge benefits and lack of negative effects -Introducing GM maize was definitely the way forward France: Thierry de l'Escaille, European Landowners' Organization, - “wide-scale adoption of these three biotech crops in Europe could significantly increase annual production, improve farmer income by more than 1 billion Euros (US$1.18 billion) and reduce spraying practices. With results like these, it's easy to understand why farmers want access to this new technology," said l'Escaille Romania: Buzdugan farmers reported price premiums of up to 10 percent for biotech soybeans due to fewer weed impurities. Average price gain 2%. Production gain K M tonnes (16-19% ) Earnings increase 35 million and 62.4 million euros (2003). I can tell you that soybean farmers in Romania are very interested in biotech seeds," “Although the seeds are 10 to 15 percent more expensive, the income gains make the extra cost more than worthwhile”

10 Blight-resistant potato
Transgenic breeding lines preferred because resistance can be introduced into commercial lines with greater speed. The BASF GM potatoes involves the use of two broad spectrum resistance genes, Rpi-blb1 and Rpi-blb2. These two genes have a structure associated with regulatory genes called nucleotide binding site-leucine rich repeat (NBS-LRR) class of regulatory proteins. Many disease resistance genes code for proteins of that class. Numerous plant NBS-LRR genes are present in the typical plant genome, each protein is specific for a particular pathogen signaling a defense response, frequently a localized plant cell death called hypersensitive response. The C terminus of the protein containing LRR recognizes a ligand feature of a pathogen activating the NBS signaling module to initiate the defense response. The blight fungus suppresses the potato defense genes in sensitive plants but is thwarted by successful defense genes. Blight-resistant potato (BASF -Rpi-blb1 and Rpi-blb2 NBS-LRR) -UI study concluded for the major potato-producing regions of the world would be $4.3 billion. 10

11 Questions and Comments?

12 “Resistance” Genes natural and otherwise
Rootknot nematodes R in tomato (Mi) and (aphids). Alternate to fumigation (Williamson UC Davis) Xylella fastidiosa Diffusible signal factor (Dsf) for disrupting Xf colonization, Inhibition of Xf polygalacturonase (PG). Targeting other Xf proteins required for virulence. protein/peptide-based inhibitors of pilins and adhesins – alternate to malathion vector control Xa21 rice R gene confers resistance to several Xoo. Defense response triggered by Xo molecule, AvrXa21. Transgenic more resistant due to copy number (Ronald) Apple Fireblight (E. amylovora) -antibiotic sprays, cecropin lytic peptide analog worked well in field trials (Norelli) Apple scab (V. inaequalis) Multiple applications of fungicides used exclusively to control this disease. 'McIntosh' trees expressing either the endo- or exochitinase gene or both increased resistance to apple scab Use of apoptosis inhibition to protect plants from mycotoxin damage (Gilchrist, UC Davis) Sclerotinia-resist sunflower oxalate oxidase Pioneer wheat Zinc Fingers Dow/Sangamo

13 Natural Disease Resistance Genes Have Been Cloned
Xa21 gene has been cloned from an African rice variety and introduced into modern cultivars to confer resistance to rice leaf blight. Pam Ronald, Plant Pathology, UC Davis

14 Use of apoptosis inhibition to protect plants from mycotoxin damage
David Gilchrist, Plant pathology, UC Davis

15 David Gilchrist, Plant pathology, UC Davis

16 Rice staple food for 2.4 billion people
Fungal diseases destroy 50 million metric tons of rice per year; varieties being developed resistant to fungi - proteins with anti-fungal properties. Insects cause a 26 million tons loss of rice per year; insecticidal proteins environmentally friendly control. Viral diseases devastate 10 million tons of rice per year; Tungro virus genome transgenes defense systems. Cassava Mosaic Virus similar protection system as papaya working in Kenya Bacterial diseases cause comparable losses - transgenes such as cecropin lytic peptide basis for inbuilt resistance.

17 STRIGA Striga (Scrophulariaceae) is a genus of obligate root-parasitic
flowering plants. All of the cultivated food-crop cereals in Africa are parasitized by one or more Striga spp. Striga spp. in the savanna regions alone account for $7 billion and are detrimental to the lives of over 100 million African people. ·        Parasitic weeds: Striga gesnerioides is a major, if not the leading, biotic constraint of cowpea production. Yield losses in infested soils from %. S. gesnerioides is widely distributed in Africa from Morocco and Sudan to S. Africa and is the most common Striga species in Africa. Most impact in cowpea agriculture in the Sahelian and savanna zones of west and Central Africa from Senegal to Chad and to southern Togo and Benin. Important in Mali, Burkina Faso, Niger, Nigeria, and Cameroon. The problem has increased in importance over last decade. A close relative of Striga, Alectra, is also parasitic on cowpeas, particularly in East, Central and Southern Africa. Yield losses of 20% in Kenya, and 50% in groundnuts in Nigeria. We propose a two-fold approach to tackle this problem. Firstly, marker tags will be developed to facilitate marker-assisted selection in classical breeding. Secondly, a transgenic approach will also be pursued to reduce chemical stimulation of Striga by cowpea. Host plants release factors required by parasitic plants Striga causes massive losses to crops in Africa:control strategy to inactivate host recognition factors - John Yoder, UC Davis,

18 Striga Orobanche Triphysaria
Host plants release factors required by parasitic plants: control strategy to inactivate host recognition factors Host plants Parasitic plants maize cowpea Arabidopsis Striga Orobanche Triphysaria sorgoleone Seed germination 0 h 12 h 24 h Haustorium development DMBQ

19 Abiotic Stress: Drought, ColdHeat, Salinity
Abiotic stress limiting factor for crops reaching genetic potential Improved water conservation -Fewer crop losses -Higher yields on all acres through improved water utilization -Expand in drylands - Nuclear Factor Y B subunit Crops limited by salinity on 40 % world's irrigated land (25 % US) Cold: Engineering with COR15a Tf, role in freezing tolerance. Plants engineered with Choline oxidase (codA) soil tolerated saline and cold Homeodomain-leucine zipper (HD-Zip) transcription factors respond to H2O & osmotic stress, exongenous abscisic acid Transport protein. Grow and fruit even in irrigation water that is > 50X saltier than normal. > 1/3 salty as seawater. Blumwald and Zhang) Abiotic Stress: Drought, ColdHeat, Salinity + Gene D Control Drought Stressed Rice + Gene E Control Grower Benefits: Improved stress tolerance allows earlier planting and further reduces yield variability and grower financial risk 70-80 million acres in US suffer yield losses due to moderate water stress. The most critical time for water stress is near pollination and flowering. e.g. yields with or without irrigation can vary 100%. Dry land acres (Kansas) can yield less than 100 bu/a without irrigation and 200 bu/acre with irrigation. About 15% of US corn acres are irrigated. A lot more acres would grow corn if yields were higher due to drought tolerance. About 20 million acres in US would benefit from a drought tolerance gene that gives a 10% yield increase. An additional 5 million acres could move from some other crop to corn if we had drought tolerance. On these new acres we would capture 100% of new units sold and associated trait fees etc. Science: Gene C source is transcription factor from arabidopsis. Found in arabidopsis screens. In the process of moving this gene into crops. Gene D is a different gene from microbial sources which confers broad stress tolerance to cold, heat and drought. What we show here is drought tolerance. Water availability and temperature stress are major causes of yield loss We use our Genomics platform to screen for genes which reduce yield loss under these conditions The bottom right pictures show target genes in Rice plants under common stress conditions 19

20 Increased Yields Yield Gene Control
- Improve Nitrogen Assimilation - Increase Sucrose hydrolysis, Starch biosynthesis - Increase O2 availability - Modify photosynthesis WHY BIOTECH IS GOOD NEWS FOR THE FERTILIZER INDUSTRY We have an active research program in yield enhancement for wheat, soybeans and corn which will be new agronomic traits. This is wheat in this picture. The larger plants on the left of the screen contain a yield gene. These wheat plants have the ability to use nitrogen better -- there is a greater spike weight (biomass) which means more yield. Use of biotechnology and genomics will contribute to this potential breakthrough. Let’s look at the benefits that improved nitrogen assimilation would bring to the industry … to the environment ... This short-day sorghum plant was used to map the Ma-1 gene (genes which modify photoperiodic behavior and thus maturity). This gene which works in other cereals would offer particular benefits to biomass and forage crops in which flowering is undesirable Yield Gene Control

21 BioFuels LDC 30 % global energy. Growth driven by population and economic . Of the world's 47 poorest countries, 38 are net oil importers, and 25 import all of their oil, consuming much of their national income to pay for it. The challenge: 5-10 times more efficient $5/gal c/gal Biomass Conversion: Organic polymeric material, lignin, starches celluloses bioconverted ethanol; hemicellulose hydrolyzed to sugars, xylose , glucose . Modify Plants and algae to improve enzymatic conversion. Modify enzymes to improve conversion and fermentation Maize other cereals, Switch grass, elephant grass poplars Biodiesel is biodegradable and non-toxic - alkyl esters made from the transesterification of vegetable oils or animal fats., (60% less CO2) Rapeseed, Botryococcus braunii (Bb) colloidal microalgae Concerns: Food trade off – Efficiency of production – ecological impact 21

22 Seeing the wood for the Trees
Agronomic: HT, Disease resistance : increased productivity reduction of plantation establishment , reduced tree losses Poplar, Aspen, Pine, Walnut, Cottonwood trialed 57% timber Cell Cycle: LFY gene, PTFL gene from Populus able to induce early flowering in poplar. Phytohormones increase size, biomass, wood quality. GA 20-oxidase (AT) aspen fast growth (D/H) increas biomass (Eriksson) Paper: low lignin, faster growing ( Sederoff/Chiang) Biofuels: low lignin (ArborGen), faster growing Concerns: robustness/health, gene flow, longevity, similar trees. China lost track of Bt poplars. Male Sterility/Flowering inhibition: restrict gene flow, grow faster and produce more wood, since energy not wasted reproducing Tapetum barnase gene prevent pollen development promoters could lead to damage of non-reproductive tissue, (long generation times of conifers are considered). RNAi down regulation (full suppression often difficult) Tissue specific cytotoxic stilbene synthase (STS) competes chalcone synthase (CHS). Somatic cells STS no competition resveratrol anti-fungal (Höfig et al. 2006) 22

23 Phytoremediation Simultaneously restores soil health and revegetates economical University of Georgia poplar trees with merA, from mercury-resistant bacteria. Soil-borne and selected for heavy metals. Absorb from soil, convert to a relatively inert form, and release as vapor. 10X Mercury removal Richard Meagher gene from the E. coli soybeans to Arabidopsis pumps arsenic from the soil and stores it in its leaves, Bioremediation: Wilfred Chen Riverside developing high-affinity microbial bioadsorbents for heavy metal removal using engineered E. coli with surface-expressed peptide analogues (ECs) of phytochelatin - microbial bioadsorbents - removal of heavy metals - cadmium, mercury, and lead. 23

24 Modify Fruit Ripening Fruit ripening modified to Improve Quality and Reduce Postharvest Losses by altering the activity of cell wall enzymes, such as polygalacturonase, involved in softening and deterioration. The biosynthesis of ethylene, the fruit-ripening hormone, has also been blocked in several ways to delay fruit ripening. Ethylene can then be applied to induce ripening when desired, as is currently done with tomatoes and bananas. Caffeine-free coffee plants and controlled ripening coffee plants. Coffee and tea plants with improved disease resistance and tolerance of environmental stresses such as cold and drought. Controlling the ripening process will allow the farmer to both control and lengthen the harvest season.

25 Questions and Comments?


27 Modern Biology / Genomics - A new Research-Paradigm in modern Biology
Phenotype Appearance and Traits of an Organism Improved Crop Plants Genotype / Genes / DNA Inherited Information Defining an Organism Genomics = the Totality of the Information of all Genes and their Functions

28 From Genomics to Improved Crops
The 2 Phases of Biology Improved Crops Phase 2 Molecular Breeding Transgenics Reverse Genetics Forward Genetics B i o i n f o r m a t i c s Genotype Phenotype GeneARNAAProteinsAMetabolitesAOrganism DNA Sequence Map Transcriptome Proteome Metabolome Profiling New Plant Traits Genomics Platform The various genomic technologies enable the profiling of the expression on each step in the chain, from Genes up to Biological Symptoms (phenotypes). Genomics technologies enable the investigation of the totality of all expressions. Phase 1

29 Metabolic Pathways are the target for complex modifications
Genome Transcriptome Proteome Metabolome Unraveling protein collaborations could change the way pathways are manipulated for improvement of valuable traits in plants. Attempts to modify storage proteins or secondary metabolic pathways more successful than have alterations of primary and intermediary metabolism

30 Plant Metabolic Engineering Challenges
Beware the flux! High Lysine maize Photo: Fruc-1, 6-Bisphosphatase Phosphoribulokinase ↓3X ↓10X, minor photosynth rate Plastid aldolase, catalyzes reverse reaction not subject to allosteric regulate, signif ↓↓ in photosynth rate & C partitioning (Haake et al., 1998) Mod storage proteins or 2nd metabolic paths > success than alt 1ry and intermed metabolism (ILSI, 2004/ 2008). Enzymes and Intermediaries known- Little known on controls and integration Metabolic Pathway Engineering Best: Mod single genes or series ind enzy. steps. Conversion of exist comp to another rather than change flux through path ID rate-limiting steps in synthesis Target to channel metab flow into new paths, Gene-silencing reduce or eliminate undesirable comps, traits, or switch off genes to inc desirable Direct DNA cassette Paul Christou 2009 Transcription Factors! Maize C1 and R, reg flavonoids aleurone , accumul rate anthocyanins activating the entire path (Bruce al 2000) Wheat Rescue ancient TFs NAC (Uauy 2006) . 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 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). Other Considerations Site of synthesis Site of activity Charge and size Redundancy 30

31 Improved Nutritional Content
Many common food crops not perfect for nutritional requirements of humans or animals. Proteins: Maize, wheat, Sweet potato and cassava WHO: 800 million people suffer from malnutrition, Protein-energy malnutrition (PEM), the most lethal form, affects 1 in 4 children: 150 M (26.7%) underweight; 182 M stunted. 70% live in Asia, 26% Africa, 4% Latin America, Caribbean Grains low in Lysine – LDCs food - Feed Rations/pollution High Lysine maize: Use non feedback- enzyme (5X ppm) N assimilation modified pathway GDH 12% increase protein SRP Nonallergenic Amaranthus Albumin for potato High Protein: Cytokinin rescue flower pair kernels fused single kernel two embryos - high protein/oil low CHO NAC Tfs (NAM) senescence and nutrient remobilization leaves to grains, RNAi delay senes 30% protein, Zn, Fe Artificial Proteins: ASP-1-sweet potato 67% increase protein (EAA 80%) 31

32 Improved Nutritional Content
Carbohydrates Starch High Amylose (resistant starch) inhibit 2 SBE Sorbitol role in fruit carbon metabolism and affects quality attributes sugar-acid balance and starch accum Wheat puroindoline genes in rice better starch/flour Fibre – Humans increase Polymers, Inulins, Fructo-oligosaccharides (FOS) SC Fructans sucrose taste: GI Tract health- fermented colonic – bifidobacteria (compete pathogenic bacteria) SC Fatty acid – anticancer/ inhibit HMG-CoAR less LDL SC fructans 1-SST Jerusalem artichoke. 90% sucrose converted "fructan beets“ (Koops, 2000) Potato synthesize the full spectrum of inulins from globe artichoke roots Lignans: enterodiol/lactone estrogen-dependent cancer Fibre – Animals Decrease Brown midrib (COMT)–Decreased lignin increase digestibility better feed conversion, livestock prefer (Sorghum) 32

33 Improved Nutritional Content
Oils and Fatty acids Altering chain length and saturation level Novel genes to produce unusual fatty acids in oilseed MUFA: High Oleic Acid: more stable than PUFA heat/ oxidation resistant, little or no postrefining (hydrogenation): AS oleate desaturase soybean gave >80% oleic acid (23%), Less SF milk/meat of animals MCT: medical foods, ergogenic aids. Acyl-ACPT canola, increase in capric (C10) and caprylic (C8) High-CLA: Antiox- free radicals heart disease/cancer Omega -3 DHA-EPA “Fish Oil” CV/thrombosis/ Cancer/ Arthritis/ Cognitive/Mental/ premies D6 Desaturase: Canola/soybean precursor SDA 3.6X ALA in generating EPA GLA safflower oil (C18:3n-6) anti-inflammatory effect, improved skin health and maintaining weight loss Sitostanol: phytosterol phospholipid Block cholesterol 33

34 Slide courtesy of Bruce Chassy

35 Improved Nutritional Content
(bacteria) (daffodil) Introduced enzyme (maize) Micro nutrients Vitamins/ minerals: Vit A Golden rice II b-carotene-Rice 25X (CBP) Biofortified cassva flour- Field trial Nigeria (Sayre) Vit B Folate increase in rice (pregnancy deficinicies) Vit E a-tocopherol g-TMT; Vit C increase corn DHAR Minerals: Ferritin (bean S protein), Metallothionein (Rice, wheat). Ca/proton antiporter (sCAX) Ca transport into vacuoles. Ca-fortified carrots enhanced absorption. Multi vitamin Corn Combinatorial direct DNA transformation rapid production of multi-complex metabolic pathways transferred 5 constructs controlled by different endosperm-specific promoters into white maize. Different enzyme combinations show distinct metabolic phenotypes – resulting in 169X beta carotene (60 mg/g v. 14 by breeding) 6X vitamin C, and 2X folate (Christou, 2009) 35

36 Improved Nutritional Content
Functional components - greater than nutrient value alone Phytochemicals: Carotenoids: Golden Rice, Sweet Potato, Cassava - (sight, development) Lycopene: polyamine Tomato – (reduce LDL, cancer) Isoflavones: genistein and daidzein; Isothiocyanates Phenolics: resveratrol antioxidant Sirtuins (anti-aging) polyphenol oxidase : help sequester protein during ensiling, Gallic acid hydrolyzable tannins, sequester protein in the rumen, more efficiently absorption Flavanols: Catechins, Flavones: quercetin (less adjuncts) Anti-nutrients: Trypsin Inhibitors; oxalic acid; furans; Phytate, Bioavailability Phosphate, divalent ions: Phytase (Rice, alfalfa) Allergens: soy P34 removal; peanut; gluten digestion Toxins: Glycoalkaloid (potato) AS solanine Cyanogenic glucoside (cassava) hydroxynitrile lyase 36

37 Increased b-Carotene in Rice Grains
Over 120 million children worldwide are deficient in vitamin A. Rice engineered to for b-carotene, converted to vit A in the body. This trait in rice cultivars distributed worldwide could prevent 1 to 2 M deaths each year. Golden Rice 23X rice 1. (daffodil) Introduced enzyme (source) Normal rice (maize) (bacteria) Seeds Phytoene Synthase Canola Carotenoids are Elevated 40 Times Oil “Golden” rice (daffodil) Ferritin, an iron-rich bean storage protein, Phytase, an enzyme that breaks down phytate making Fe available, reabsorption of iron, a gene for a cystein-rich metallothionein-like protein has been engineered into rice “Golden” rice 2 UCDAVIS Ye et al. (2000) Science 287: A lack of dietary iron, zinc and calcium results in unhealthy increases in cadmium uptake into the kidney and liver

38 Questions and Comments?

39 Nutrigenomics Nutritional Genomics
"Leave your drugs in the chemist's pot if you can heal the patient with food." (Hippocrates) Nutrigenomics refers to the prospective analysis of differences among nutrients in the regulation of gene expression Nutrigenomics/Nutritional Genomics/ Nutrigenetics?: Genetically based, nutrition intervention that maximizes the health and effectiveness of each individual. Monogenic Diseases: 97% of the “disease-associated” genes Phenylketonuria, phenylalanine hydroxylase PHE -> TYR. Leads to neurological damage and mental retardation. PHE restricted TYR-supplemented diets -no Aspartame! Lactose Intolerance – Juvenile enzyme active into adulthood Polygenic diseases obesity, cancer, diabetes, and cardiovascular diseases, SNP in haemochromatosis linked gene (HFE) risk, MTHFR PM higher intakes of folic acid serum homocysteine Most dietary effect specific interactions on molecular level, regulation of gene expression directly and indirectly activity of transcription factors

40 Nancy Fogg-Johnson: Nutrigenomics will revolutionize health and nutrition – It will inform how we prevent and treat disease and how food is grown, processed, and made. Eventually nutrigenomics will be able to discover diets that prevent or retard the onset of the most serious and widespread of today's killer diseases, like cancer, as well as degenerative diseases like Alzheimer's. When/if?

41 Sitosterolemia (hyperabsorption of sterols hypercholesterolemia risk for atherosclerosis). Regulation of sterol uptake Mice was treated with a lipid metabolism-altering drug DNA microarray used for expression profiling of various tissues. Differential display with a control led to the discovery of an unknown gene. Computer simulation found that two proteins gene regulated reverse transport of dietary sterols out of the apical surface of intestinal cells. Exploring human gene databases, found a human homologue This explained why dietary sterols, which are structurally similar to cholesterol, are not absorbed in normal individuals. By scanning sitosterolemic individuals for this gene, it was found that all of them had a mutation in this gene responsible for their uncontrolled hyperabsorption of dietary sterols.

42 Nutrigenomics Nutritional Genomics
Number of genes regulate lipid metabolism/insulin sensitivity, affect susceptibility to T2 diabetes mellitus. SREBP-1c (sterol response element binding protein) mutations led to fatty livers, hypertriglyceridemia, severe insulin resistance, type 2 diabetes mellitus. One polymorph expression highly induced in mice on high fructose diets. Two missense mutations in exons domain of SREBP-1c were found in individuals displaying severe insulin resistance. Another association was found between an intronic single nucleotide polymorphism (C/T) between exons 18c and 19c and the onset of diabetes in men, but not in women. T2DM Asian/Hispanic populations insulin resistance rather than b-cell dysfunction. In African-Americans the opposite. Hyperlipidemia: E4 allele in the apolipoprotein E higher LDL compared with the other (E1, E2, E3) for same fat intake levels

43 Nutrigenomics Nutritional Genomics
One single nucleotide polymorphism (-75 G/A) in the apolipoprotein A1 gene in women is associated with an increase in HDL . ApoA1 women showed increase in HDL with increase in PUFA compared to G variant taking similar amounts of PUFA. Haplotype (HapK) in leukotriene A4 hydrolase (LTA4H) risk of myocardial infarction (MI) European and African Americans. MI significantly greater in African-Americans HapK. (n6/n3) Barbecue Heterocyclic aromatic amines acetylated to reactive metabolites bind DNA - colon cancers. N-Acetyltransferase NAT is a phase II metabolism enzyme that exists in two forms: NAT1 and NAT2. HAA can be activated through acetylation to reactive metabolites which bind DNA and cause cancers. Only NAT2 fast acetylators can perform this acetylation. Studies have shown that the NAT2 fast acetylator genotype had a higher risk of developing colon cancer in people who consumed relatively large quantities of red meat.

44 Caveat Emptor! Will there be implications for your insurance if you have a susceptibility to heart disease? Will there be implications if you fail to follow a diet to retard the onset of symptoms? "Nutrigenetic Testing: Tests Purchased from Web Sites Mislead Consumers.“ Government Accountability Office (GAO) commercial "nutrigenetic" testing dubious clinical validity of commercial genetic tests, and unethical practices Investigators posed as 14 clients used the DNA from just 2 - man (48)-girl (9mnth). Despite this, the test 'results' were contradictory and warned of risks for various conditions. osteoporosis, heart disease, diabetes and more. Affiliated companies then offer nutritional supplements to stave off these predicted sicknesses — but the pills turn out to be little more than multivitamins, offered with a hefty dose of misleading medical advice. Cost to you a mere $89 to $395! (Nature, 2006)

45 How to Produce a Bio-therapeutic
Glycosylation Folding Volume System Comments Complexity required System well understood unproven Appropriate for Mabs, fusion proteins Volume> 100 KGs Transgenic Animals HIGH Protein Complexity Well characterized System (Mammalian) Limited capacity Opt Manufacturing strategy Volume< 100 KGs Cell Culture (mammalian, Insect, plant) YES LOW Glycosylation Required Volume> 100 KGs Transgenic Plants System early unproven Appropriate for Mabs, new direction HIGH NO Protein Complexity Volume<100 KGs Microbes Well characterized System Excess capacity LOW

46 The Cow Pock or the Wonderful Effects of the New Inoculation
The Cow Pock or the Wonderful Effects of the New Inoculation! James Gillray ( ) Vide--The Publications of ye Anti-Vaccine Society, June 12, 1802, Library of Medicine Pasteurization

47 Vaccines create antibodies to neutralize the causative virus, bacteria or toxin.Antigens may be introduced by:Killed Vaccines Killed vaccines designed to create antibodies without the negative effects of infection so are generally considered to be safe. However, during the inactivation process, some of the surface antigens needed to create the desired antibodies may be destroyed thus reducing their effectiveness. Modified live vaccines contain an attenuated or weakened version of a disease agent. Modified live vaccines are effective but can negatively impact the health of the animal. Subunit vaccines use only the necessary parts of the virus to stimulate immunity. Unlike modified live vaccines, subunit vaccines stimulate the immune system to prevent disease without stressing the animal. And unlike killed vaccines, subunit vaccines do a better job of disease prevention as they only contain concentrated amounts of the target antigen. These qualities make subunit vaccines both safe and effective. Plant-made vaccines are a new type of subunit vaccines.


49 Rinder Pest Devastating Disease 100% Fatal
Existing Vaccine: Problems transport, lack of refrigeration, and lack of a simple system for administration. Recombinant product, freeze-dried, abating problems with transportation and handling, administered to scarified skin to regenerate the serum. The vaccinia virus is attenuated, The recombinant vaccine - two of the viral surface antigens H and F eliminates risk of contracting the disease it is easy to determine if the animal has been vaccinated and is not just a survivor. Vaccination of cattle results in a high level of immunity, affording protection against test inoculations of 1000 times the lethal dose of rinderpest virus. Tilahun Yilma, UC Davis

50 Why Make Pharmaceuticals in Plants?
Supply the increasing demand for new biotech drugs (esp. antibodies) 50 Mabs by 2010 Significantly decrease unit costs Improve patients’ access to biotech medicines Plants are an efficient producer of proteins Plants are scalable bioreactors Plants provide cost advantages to mammalian cell culture systems 3-5 times faster than mammalian systems Plant cells are similar to human cells Similar protein synthesis machinery Read the same genetic code Assemble, fold and secrete complex proteins

51 Why Plants? Produce protein ( primarily Mabs)
Correct post-translational modification mostly (unlike Microbes) No propagation of human pathogens or other mammalian contaminants; no other mammalian contaminants de novo; Asepsis can begin at purification, not inoculation (less go wrong) Scale-up utilizes the same technology used in agriculture to-day. Faster, cheaper, more convent, more efficient than CHO cells Why Seeds? Protein at the highest levels in the harvestable seed. Seeds are easier, more economical than whole plants to transport to a processing factory Proteins can be extracted and purified in prep. for packaging. Seeds can be stored for prolonged periods with protein intact. Hundreds of acres of protein-containing seeds could inexpensively double the production CHO bioreactor factory.

52 Plants as Chemical Factories
Malting grain model (Ventria, Inc., CA and defunct attempt in MO, now Kansas) Transform rice with desired protein controlled by a-amylase promoter in the aleurone layer. proteins lactoferrin and lysozyme Imbibe seeds to induce expression of a-amylase promoter & production of desired protein. Extract and purify proteins from germinating seeds. Grow rice crop in the field, harvest grain at maturity. imbibe = have seed take up water to induce germination Rice Lactoferin Lysozyme Peru 30% Less Diarrhea, Quicker recovery 3/6 days, /3 less recurrence Ray Rodriguez, Molecular and Cellular Biology, UC Davis

53 (C) 2002 JAIC/Davis Bioscience Fund
2017年4月8日 Plant Based Expression Systems for Efficient Production of Human Therapeutics KAREN A. McDONALD/ BRYCE FALK Fellow: MICHAEL A. PLESHA Human deficiency in alpha-1-antitrypsin (AAT) results in non-smoking related emphysema AAT expression kinetics via agroinfiltration of Nicotiana benthamiana plants will be optimized AAT is used as a model human protein; other proteins will also be expressed and optimized Alternate scalable induction methods will be evaluated The optimized human AAT gene has been cloned into Agrobacterium and is expressed using a chemically inducible plant viral (CMV) replicon expression system 1a 2a H AAT RNA 3a RNA AAT CMV 2a CMV 2b Promoter CMV 3a Inducible Promoter CMV 1a Replicase Inducer ,

54 Genetic Engineering Technology Allows Production of Novel Products
Passive Vaccines Ab enteric bacteria E.coli O157:H7 meat foodborne path Metabolic Pathways Viruses PlantAb protected mice against genital herpes similar physical props to MCC remained stable in human exhibited no diff in affinity for binding, neutralizing HSV Polyhydroxybuterate biodegradable plastic Acetylenic &Vernolic Acid Containing Chemical feedstocks ANTIBODY-CONTAINING SOYBEANS During the early 1990s, researchers discovered that special vaccination of flocks of chickens caused them to secrete antibodies [against the bacterial strains chosen for vaccination] into the whites of the eggs they laid. Those egg whites are now chopped-up to prepare a commercial piglet feed that removes all E. coli bacteria of specific diarrhea-causing strains from the intestines of piglets. xx That egg-white-based feed product works via each antibody “latching onto” one of the diarrhea-strain-specific E. coli bacteria within the piglet’s digestive system, then the combined pair is excreted by the animal. Because antibodies are pure protein molecules, there are no regulatory issues pertaining to meat residues. Today’s periodic outbreaks of beef-borne E. coli 0157:H7 bacterial disease occur because cattle became tolerant of E. coli 0157:H7 in the 1970s [it had previously killed infected cattle] xxi and humans are now sometimes exposed to that deadly bacteria when the hide or digestive system contents of cattle come into contact with meat (e.g., at slaughterhouses). xxii A study published by USDA in April, 2000 showed that reducing E. coli 0157:H7 in live cattle prior to slaughter greatly increases slaughterplant safety. xxii It is now possible for biotechnology to cause specific antibodies (e.g., specific to E. coli 0157:H7) to be produced in soybeans, so such future soybeans could be fed to livestock for 72 hours prior to slaughter in order to eliminate outbreaks of foodborne diseases such as E. coli 0157:H7, Salmonella spp, etc. 12. “VACCINE-CONTAINING” SOYBEANS AND MAIZE During 2001, a U.S. company will introduce a biotechnology derived maize that produces antigens for the swine disease known as transmissible gastroenteritis virus (TGEV). When that maize is eaten and those antigens in it tough lymph tissues in the swine’ digestive system, the animals’ immune system rapidly produces antibodies that protect it against TGEV. Similar plant vaccines are expected in the future for human diseases such as hepatitis B. Medical Benefits Plants have been a valuable source of pharmaceuticals for centuries. During the past decade, however, intensive research has focused on expanding this source through rDNA biotechnology. The re-search brings closer to reality the pros-pect of commercial production in plants of edible vaccines and therapeutics for preventing and treating animal and hu-man diseases. Possibilities include a wide variety of compounds, ranging from vac-cine antigens against hepatitis B and Nor-walk viruses (Arntzen, 1997; Dixon and Arntzen, 1997; Mason et al., 1992, 1998) and Pseudomonas aeruginosa and Staphy-lococcus aureus (Brennan et al., 1999) to vaccines against cancer and diabetes. In addition, genetically modified strains of probiotic microorganisms are also possi-ble vehicles for successful delivery of vac-cines and digestive aids (e.g., lactase) through the stomach and the small intes-tine. Two seminal papers supported the use of rDNA biotechnology-derived plants for pharmaceutical production (Ma et al., 1995, 1997). These reports were soon followed by one (Ma et al., 1998) describing results of successful hu-man clinical trials with an edible vaccine against a pathogenic strain of E. coli and a monoclonal antibody against cariogenic Streptococcus mutans. Haq et al. (1995) reported the expression in potato plants of a vaccine against E. coli enterotoxin against the toxin in mice. Human clinical trials suggest that oral vaccination against either of the closely related enterotoxins of Vibrio cholerae and E. coli induces pro-duction of antibodies that can neutralize the respective toxins by preventing them from binding to gut cells. Ma et al. (1995, 1998) showed that tobacco plants could express secretory antibodies or “planti-bodies” against the cell surface adhesion protein of S. mutans. Used as a bactericid-al mouthwash, the antibodies prevented bacterial colonization by the microorgan-ism and development of dental caries for four months. A similar approach showed that soy-bean- produced antibodies protected mice against infection by genital herpes (Zeit-lin et al., 1998). Compared to antibodies produced in mammalian cell culture, the plantibodies had similar physical proper-ties, remained stable in human reproduc-tive fluids, and exhibited no differences in their affinity for binding and neutralizing herpes simplex virus. Hence, the differ-ence in the glycosylation processes of plants and animals does not appear to af-fect the immune functions of the plant-derived antibodies. Non-Hodgkins B-cell lymphoma, the most widespread cancer of the lymph sys-tem, is difficult to treat because the B-cell tumors are variable and response to treat-ment can vary from person to person. Hence, effective therapy requires “person-alized medicine” tailored to the genetic makeup of each patient’s tumor. Unfortu-nately, conventional treatment methods do not meet the needs for rapid produc-tion of customized antibodies in suffi-cient quantities. Monoclonal antibodies used in conventional treatment also tend to be expensive and unreliable, and those produced in bacteria have solubility and conformation problems. A system using tobacco mosaic virus (TMV) was developed to produce in to-bacco plants (Nicotiana benthamiana) a therapeutic vaccine against non-Hodgkin’s B-cell lymphoma in a mouse model (McCormick et al., 1999). Using cells cloned from malignant B-cells of mice, TMV DNA was modified with a tu-mor- specific sequence from the gene cod-ing for the immunoglobin cell surface marker. Plants were then infected with the modified virus, resulting in expres-sion of cancer-specific antibodies. B-cell proteins were then extracted from the plant leaves for vaccination of the mice. Eighty percent of the mice receiving the while all untreated mice died within three weeks of contracting the dis-ease. A similar approach was used to devel-op a vaccine against insulin-dependent diabetes mellitus (IDDM), an auto-im-mune disease in which insulin-producing cells of the pancreas are destroyed by the cytotoxic T lymphocytes. The “oral toler-ance” method of preventing or delaying autoimmune disease symptoms involves the ingestion of large amounts of immu-nogenic proteins that turn off the auto-immune response. This method of vacci-nation is gaining recognition as a poten-tial alternative to systemic drug therapy, which is often ineffective. Insulin and pancreatic glutamic acid decarboxylase (GAD), which are linked to the onset of IDDM, are candidates for use as oral vac-cines. Blanas et al. (1996) described the development in a mouse model of a po-tato- based insulin vaccine that is almost 100 times more powerful than the exist-ing vaccine in preventing IDDM. Feeding diabetes-prone mice potatoes engineered to produce immunogenic GAD reduced the incidence of disease and immune re-sponse severity. rDNA biotechnology-derived vac-cines are potentially cheap, convenient to distribute, and simple and safe to ad-minister. Production of medically im-portant substances via rDNA biotech-nology engineering of plants and micro-organisms offers multiple advantages. For plants, production can be done vir-tually anywhere and has the potential to address problems associated with deliv-ery of vaccines to people in developing countries. Products from these alterna-tive sources do not require a so-called “cold chain” of refrigerated transport and storage, although they will require segregation from conventional foods to prevent inappropriate consumption. Pharmaceuticals or therapeutics pro-duced via genetic engineering of plants also offer an alternative delivery meth-od, feeding versus injection (Howard, 1999), and an alternative to extraction from animal sources. Furthermore, rDNA biotechnology-derived vaccines may also be safer than many conven-tional vaccines because they consist of pathogen or antibody subunits rather than whole microorganisms. The use of plants can facilitate abundant produc-tion of therapeutic proteins without the risk of contamination by animal patho-gens, and at substantially reduced cost. Biotechnology Report: Benefits C O N T I N U E D Active Vaccines Transmissible gastroenteritis virus Antigen Cholera/Hep B/banana

55 The Dow AgroSciences unique Concert™ Plant-Cell-Produced System uses plant cells, rather than whole plants, in a culture medium comprised primarily of water, sugar and salt. This eliminates the possibility of contamination and environment concerns. The Concert™ Plant-Cell-Produced System is totally bio-contained as all production is done in a sealed and sterile production process. This means only the required inputs get into the vaccine and only a safe, non-replicating vaccine comes out.

56 Alfalfa Plasma Proteins, Foot-and-mouth disease vaccine Maize Anti- HIV and Anti herpes Simplex Antibodies Microbiocides for pulmonary infection Mabs for cancer, autoimmune disease RA, Vaccines hepatitis B, Norwalk virus (Travelers disease), Vaccines & Mabs for animal, Aprotinin for blood loss, Gastric lipase cystic fibrosis, Lettuce Vaccines for Hepatitis B Moss Factor IX for hemophilia B Rice Lactoferrin Lysozyme for GI health, Alternatives to abs in poultry diets, Topical infections, inflammations, B-cell lymphoma vaccine Safflower Therapeutics and oil-based products for oral/dermal delivery Spinach Protective antigen for vaccine against Bacillus anthracis Soybean Tobacco extensin signal peptide - Anti-HSV-2 (IgG) Tobacco Non-Hodgkins B-cell lymphoma, TGF-b glucocerebrosidase for Gauchers Syndrome , Alpha galactosidase for enzyme replacement therapy, IgGs for prevention of dental decay, common cold, GAD 7 cytokines for type 1 Diabetes, Colon cancer surface antigen – Fabrazyme fat-storage disorder Tomato , Potato Banana (someday!) Edible vaccines: Enterotoxigenic E. coli, Norwalk virus, Hepatitis B, Vibrio cholera, Rabies virus-intact Glycoprotein Antimicrobe peptides, Wheat Carcinoembryonic antigen - Murine IgG signal peptide Potato Polyhydroxybuterate biodegradable plastic

57 Concerns? potential gene flow to food crops of the same species
co-mingling of food and non- food crops worker exposure to plant material containing active pharmaceutical ingredients (APIs). ProdiGene paid $3 million bond for violations in Nebraska and Iowa reimburse the USDA for the costs, involved in disposing of the contaminated crops. Now making brazzein ( a sweet protein) in maize!

58 Principles of Confinement
Physical and biological Confinement essentially means keeping the crop and its products on the land where it was grown until removed for processing, with no inadvertent exposure to the public and minimal exposure of products to workers and the environment. Identity Preservation: A Closed Loop System Preventing co-mingling or gene flow prime directive for industry as well as regulatory agencies. (No StarLink®, repetition!).

59 Concerns/Confinement
Potential gene flow to food crops of the same species Vectors: Birds, insects, viruses Co-mingling of food and non-food crops Worker exposure active pharmaceutical ingredients (APIs). Identity Preservation: A Closed Loop System Preventing co-mingling or gene flow prime directive for industry as well as regulatory agencies. Physical and biological Confinement: physical, temporal and biological isolation, as well as appropriate spatial isolation, acreage limitations, dedicated equipment, facilities “Trap” Plant borders, Color coded kernels, Male sterile plants, work well - limited to a few species. Transformation plastid (not all pollen is plastid-free, illegit recomb none of the transgenes functional in nuclear genome) Genetic Use Restriction Technologies, or GURTs

60 Depending on the timing, expression of the lethal gene leads either to seeds that are incapable of germinating or to death of the seedling. During seed maturation stage lea promoters ( “late embryogenesis abundant”) activate the genes whose products control and accompany the drying process. Shortly after the seeds have swollen, germination-specific promoters activate the genes whose products break down substances from the storage tissue and transport them to the young seedling. T- GURT seed will germinate only when treated with this inductor chemical. V-GURT seed treated with an inductor that activates the lethal gene. Produce seeds that mature but that are no longer able to germinate and cannot therefore be used for re-sowing.

61 Questions and Comments?

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