INFORMATION SYSTEMS FOR BIOTECHNOLOGY


June 2007
COVERING AGRICULTURAL AND ENVIRONMENTAL BIOTECHNOLOGY DEVELOPMENTS


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IN THIS ISSUE:



GENE-DELETOR: A NEW TOOL TO ADDRESS CONCERNS OVER GE CROPS
Yi Li, Hui Duan, and William Smith

The gene-deletor technology
Traditionally, site-specific DNA recombination systems, such as bacterial phage Cre/loxP and Saccharomyces cerevisiae FLP/FRT systems, have been used to excise marker genes or short spacer sequences in higher plants1. FLP/FRT and Cre/loxP function through interactions of a recombinase with its specific recognition sites. Recombinase recognizes these flanking recognition sites and excises any intervening DNA.

The laboratory of Dr. Yi Li, University of Connecticut, has recently developed a new technology called "gene-deletor" or "GM-gene-deletor"3 (see: http://gene-deletor.net). The principle of the technology is shown in Figure 1. As shown in tobacco, the gene-deletor technology, with a loxP-FRT hybrid as the recognition sequences for FLP or Cre recombinase, is 100% efficient in deleting all functional transgenes from both pollen and seeds, based on examination of more than 25,000 progeny per transgenic event.

The flow of transgenes into the wild via pollen and seeds is a serious concern for both the public and the scientific communities1. There are also concerns that novel proteins in certain transgenic crops could cause adverse reactions in some individuals. The gene-deletor technology could be used to remove all transgenes from any organs of a transgenic plant when the functions of transgenes are no longer needed or their presence may cause concerns. The technology may provide a useful tool to address transgene flow problems and also food safety concerns over transgenic crops.

Prevention of gene flow from asexually propagated transgenic crops
The current version of the gene-deletor technology (Fig. 1) is readily applicable to address the pollen- and seed-mediated transgene flow problems of vegetatively propagated plants that are important to the agricultural, forestry, bio-energy, ornamental, and paper/pulping industries. This technology can also be used in corn, soybean, cotton, or rice if artificial seeds (i.e., no pollination or fertilization is needed) are used for the propagation of these crops. Artificial seeds have been produced from many major agricultural crops.

Figure 1. A schematic illustration of the principle of the gene-deletor technology for excision of all transgenes from pollen and seeds. L represents the LoxP recognition sequence from the phage Cre/loxP system and F represents the FRT recognition sequence of the yeast FLP/FRT system. PAB5 is a pollen- and seed-specific gene promoter from Arabidopsis and FLP is a DNA recombinase from the FRT/FLP system. Expression of recombinase under the control of PAB5 leads to deletion of all functional transgenes between the two LF (LoxP-FRT fusion) sites, including the recombinase gene in pollen and seeds specifically. The deleted gene sequences will be destroyed by non-specific nucleases in the cell. Because of the use of the LoxP-FRT fusion as the recognition sequence for FLP, the deletion efficiency observed is as high as 100%, based on more than 25,000 T1 progeny examined per event.

 

Prevention of gene flow from sexually propagated transgenic crops
A modified version of the current gene-deletor technology could also be used in sexually propagated crops such as corn, rice, soybean, cotton, sunflower, canola, and sorghum, etc. One possible strategy is to introduce a chemically inducible RNAi-FLP gene cassette into the gene-deletor system. Application of a chemical inducer at the correct time would prevent the deletion of transgenes in pollen and seeds (Fig. 2). For instance, the ethanol inducible ALCR/alcA (alc) two-component system4 could be used to control RNAi-FLP gene expression. With the ALCR/alcA (alc) system, application of ethanol during pollen/seed development would activate the RNAi-FLP gene. RNAi-FLP represses the pollen/seed specific expression of FLP recombinase. As a result, all transgenes will remain in the pollen or seeds during that generation. However, if the inducer is not applied in a subsequent generation, FLP will be expressed. Consequently, FLP expression will lead to deletion of all functional transgenes in pollen and seeds.

Figure 2. The principle of a modified version of the current gene-deletor system for its applications in sexually propagated crops. FLP is a DNA recombinase that recognizes the FRT sequences and excises the intervening DNA sequences. PAB5 is a pollen- and seed-specific gene promoter. If an inducible RNAi-FLP gene cassette is inserted in the gene-deletor system, application of the inducer will activate RNAi-FLP expression and therefore suppress FLP expression. If the expression of FLP is suppressed, deletion of transgenes will not occur. During the sexual production of certified seeds, seed companies could apply the inducer to prevent the deletion of transgenes from pollen and seeds as shown. In a subsequent generation, all transgenes will be deleted from pollen and seeds as shown in Fig. 1 if the inducer is not applied.

 

One important feature of the inducible RNAi-FLP gene is that it does not need to be 100% efficient if used in the gene-deletor system. Non-perfect suppression of FLP by RNAi-FLP could cause an undesirable deletion of transgenes in a small number of pollen and seeds. However, non-perfect suppression of FLP expression will not present a problem because seed companies can easily eliminate these "non-transgenic" seeds when producing certified seeds. Seed companies may apply a selection agent such as herbicide at early stages of seed development to kill non-transgenic seeds. Also, if a small percentage of certified seeds are non-transgenic, it should not cause a significant yield loss to farmers. For crops that are engineered for insect resistance, some researchers believe that planting 1% to 3% non-transgenic plants in an insect resistant field may reduce the potential risks for developing resistant insects. Furthermore, because application of the chemical inducer is performed by seed companies, and acreages for certified seed production are generally small, it is relatively easy to achieve uniform and effective applications of the inducer.

Alleviation of the safety concerns over transgenic foods
The gene-deletor technology can be extended to generate non-transgenic edible parts from transgenic plants to mitigate consumer concerns over the presence of transgenes in food products. As shown in Figure 3, if one uses a pollen- and seed-specific gene promoter to control recombinase expression, all transgenes will be deleted from pollen and seeds specifically. This is particularly useful for herbicide resistant crops like soybean. Similarly, with a fruit- or tuber-specific gene promoter to control FLP expression, one can produce non-transgenic fruits or tubers from transgenic crops. The technology could also be used for post-harvest removal of transgenes in crops like lettuce and broccoli with a conditionally active (e.g., post-harvesting active, high- or low- temperature inducible, or ethanol-inducible) gene promoter to control FLP expression. In all of these cases, the transgenic protein products will be destroyed in a matter of hours or days after the transgenes are deleted.

Figure 3. The gene-deletor technology can be used to produce non-transgenic organs from transgenic plants (Transgenic is shown in red; non-transgenic is shown in green). Transgenic plants produced from the current technologies contain transgenes in all organs and cells (all transgenic). With the gene-deletor technology, non-transgenic organs could be produced if an appropriate gene promoter is used to control FLP expression.

 

Prevention of transgenic volunteer crops
Volunteer crops are plants growing from seeds which fall out in harvesting from a previous planting. Volunteer transgenic plants are a source of many environmental risks and have resulted in lawsuits from neighboring farms or seed companies. Because the gene-deletor technology can remove all functional transgenes from pollen and seeds, there will be no transgenic volunteer plants in subsequent seasons.

A better technology than sterile seed technologies
Compared to sterile seed technologies like the terminator technology5, the gene-deletor technology offers several advantages. Similar to the sterile technologies, the gene-deletor technology can address the problem of pollen- and seed-mediated gene flow. However, the gene-deletor technology is superior to sterile seed technologies because it can be used to produce non-transgenic seeds, fruits, tubers, flowers, leaves, or stems to alleviate the concerns over transgenic food safety. Furthermore, unlike the sterile seed technologies that may pose potential problems for farmers in developing countries, the gene-deletor technology allows the production of viable seeds and the yield of the subsequent generation crops is the same as their non-transgenic counterparts.

The UConn team started the development of the gene-deletor technology in 2000 with funding from Connecticut Innovation Inc (CII) and from the Consortium for Plant Biotechnology Research (CPBR) and UConn. The team and their collaborators at Chinese Southwest University and the University of Tennessee published the technology in the March issue of Plant Biotechnology Journal in 2007.

References and additional readings:

1. Srivastava V, Ow DW. (2004) Marker-free site-specific gene integration in plants. Trends Biotechnol. 22(12), 627-9

2. Stewart CN Jr, Halfhill MD, Warwick SI. (2003) Transgene introgression from genetically modified crops to their wild relatives. Nat Rev Genet. 4(10), 806-17

3. Luo K, et al. (2007) 'GM-gene-deletor': Fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seeds of tobacco plants. Plant Biotech. J. 5(2), 263-274

4. Roberts GR, et al. (2005) The alc-GR system: A modified alc gene switch designed for use in plant tissue culture. Plant Physiol. 138, 1259-1267

5. Daniell H. (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol. 20(6).581-586

Gene-Deletor: For additional information, news reports and commentaries about the gene-deletor technology, please visit: http:gene-deletor.net

Yi Li
Department of Plant Science, U-5082
University of Connecticut
Storrs, CT 06268
yi.li@uconn.edu

William Smith
Department of Plant science
University of Connecticut
Storrs, CT 06268

Hui Duan
Department of Cell & Structural Biology
University of Illinois,
Urbana, IL 61801



METABOLIC ENGINEERING THROUGH PLASTID TRANSFORMATION
Tawanda Zidenga

Making a case for transplastomic plants
In plants, genetic material is distributed between the nucleus, plastids, and mitochondria. Production of transgenic plants, for basic and applied purposes, has traditionally been mainly through expression of transgenes in the nucleus. Among the concerns raised about genetically engineered organisms is the fear that transgenes may "escape," for example via pollen in corn, "contaminating" non-transformed plants. Such concerns have given birth to a new field of transgene containment. Along these lines, plant biotechnologists have been keen on expressing transgenes in chloroplasts rather than the nucleus. The advantage of these transplastomic plants is that chloroplasts usually exhibit a strict maternal inheritance, and will therefore not be found in, say, pollen grains of corn. Genes expressed in the plastome will therefore not be transferred through pollination.

Chloroplast transformation also carries with it additional advantages. First, the high ploidy number of the plastid genome, the plastome, allows for high levels of expression. Diploid cells bear two copies of the nuclear genome, while they may contain hundreds of chloroplasts. Second, transgene integration in the chloroplast occurs via homologous recombination (site-specific integration), minimizing position effects.

David Wurbs, Stephanie Ruf, and Ralph Bock of the Max Plank Institute in Germany attempted to elevate levels of pro-Vitamin A in tomato via plastid metabolic engineering1.

Engineering for increased carotenoids
Plant metabolic engineering offers many possibilities for increasing the nutritional characteristics of plants, as well as enhancing production of industrial plant products. Carotenoids, which are naturally occurring organic pigments in plants, can be grouped into two categories: xanthophylls (which contain oxygen) and carotenes (which are oxygen-free). β-carotene, also called pro-vitamin A, is the precursor to vitamin A and plays an important role in vision, growth, and development2. It is therefore an important dietary component.

The major storage carotenoid in tomato is lycopene, an antioxidant that gives tomatoes their color. In the tomato fruit, lycopene accumulates in chromoplasts, a type of plastid that differentiates from chloroplasts during fruit ripening. Lycopene can be converted into β-carotene by the lycopene β-cyclase enzyme, as indicated in Figure 1 below. This enzyme is not present in humans and other animals, which explains why we are dependent on our diets for pro-vitamin A.

Figure 1. Conversion of lycopene to either β-carotene or ά-carotene. Modified from The Plant Journal 49, 276-288.

Wurbs et al. used the bacterial lycopene β-cyclase gene to elevate carotenoid biosynthesis in tomato and reported increases in β-carotene in ripe tomato fruit, a non-green plant tissue.

The construct
The basic chloroplast transformation vector pRB96 was used. This vector harbors a transgene expression cassette within the polylinker. The cassette consists of the promoter and 5’ UTR from the plastid atpI gene and the 3’ UTR of the rps16 gene.

Figure 2. Map of the vector and the insert (lycopene β-cyclase from Erwinia LCE).   Source: The Plant Journal 49, 276-288.

Achieving homoplasmy
Heteroplasmic situations (presence of wild type copies of chloroplast genome) are considered genetically unstable4. One way to achieve homoplasmy is to allow a sufficient number of cell divisions under selection. Ultimate confirmation of homoplasmy was done by seed assays, and the lack of segregation of antibiotic resistance in the T1 generation demonstrated homoplasmy and maternal inheritance of the transgene.1

Analyzing transplastomic plants
When transforming plants to increase carotenoids, an increase of only the carotenoids is typically desired. This means that, other than the pathway being engineered, phenotypically the transgenics should resemble wild types. The researchers checked the phenotypes of the transplastomic plants and found they were similar to wild type controls, which is good. Next, they grew the plants in media containing 2-(4-chlorophenylthio)-triethylamine (CPTA), a specific inhibitor for the cyclization of lycopene. CPTA works by inhibiting the lycopene β-cyclase enzyme.

The rationale for the approach is that plants showing increased levels of lycopene β-cyclase (i.e., transplastomic plants) would exhibit increased tolerance to CPTA. Indeed, transplastomic plants tolerated CPTA, indicating that transplastomic expression of bacterial lycopene β-cyclase indeed elevates enzyme levels in the plastids of transformed plants. The increased amount of the enzyme also correlated with increased activity, and therefore increased production of β-carotene (visible as a color change of the fruit as shown in Figure 3 below). Fruits from transplastomic plants had a more orange pericarp and flesh, which could be diminished by adding CPTA

Figure 3. Phenotype of ripe tomato fruits harvested from transplastomic tomato plants expressing the lycopene-cyclase gene from Erwinia.
Top row: Wild type; Middle row: Transplastomic tomatoes expressing lycopene b-cyclase; Bottom row: Fruits from transplastomic plants treated with 2.5 mM CPTA.
  Source: The Plant Journal 49, 276-288.

Conclusions
This report by Wurbs et al. shows the extension of plastid transformation from the tobacco model plant to tomato, also an important crop plant. The demonstration of successful metabolic engineering via plastid expression of an important pathway is indeed a promising result. It also demonstrates the feasibility of using transplastomic technology for non-green tissues1. Plastid transformation has also recently been achieved in trees3.

Sources

1. The Plant Journal (2007) 49, 276–288

2. Ohio State University Extension Fact Sheet. http://ohioline.osu.edu/hyg-fact/5000/5551.html

3. Transgenic Res (2006) 15:637–646

4. J. Mol. Biol. (2001) 312, 425-438

Tawanda Zidenga
Department of Plant Cellular and Molecular Biology
The Ohio State University
zidenga.1@osu.edu


GENETICALLY ENGINEERING FOLATE BIOFORTIFICATION IN TOMATO
Claudia Lerma-Ortiz

Tetrahydrofolate (THF) and its derivatives (folates) are indispensable for the health of humans and other organisms. They participate as cofactors in one carbon transfer reactions in the synthesis of glycine, serine, methionine, purines, and thymidylate1,2. Humans are unable to synthesize folates. A deficiency of this vitamin can cause a range of serious diseases, including some birth defects (such as spina bifida), megaloblastic anemia, cardiovascular conditions, and some cancers3. Although folate deficiency is still a serious problem all over the world, it has the highest impact in developing countries, causing 200,000 severe birth defects every year.

To help prevent folate deficiency, some western countries, including the United States, have made the fortification of grain products with synthetic folic acid mandatory. Fortification helps people get the recommended allowance—400 µg/day for adults and 600 µg/day for pregnant women—in their regular diet. Unfortunately, in developing countries this kind of fortification presents a series of challenges—distribution inequities, recurrent costs, and lack of a food industry. Furthermore, fortification may itself cause some health problems. When a large amount of this vitamin is ingested, unreduced folic acid can enter into the systemic circulation4. Therefore, to achieve a greater margin of safety and lower costs, the consumption of common foods with a high content of natural THF would be a better solution.

Another consideration is that most folate in people's diets comes from plant foods. However, people regularly consume tubers, cereals, and fruits and not green leafy vegetables, which have the highest levels of this vitamin. For these reasons, a good strategy to improve folate intake worldwide would be to genetically engineer common food plants to make more folates. Hanson and Gregory’s laboratories (University of Florida’s Institute of Food and Agricultural Sciences) chose tomatoes (Solanum lycopersicum) for this experimental approach2,4. Their work is reviewed here.

Biochemistry of folates
Molecularly, folate is comprised of three parts—a p-aminobenzoate (PABA) central motif linked via its carboxyl group to one or more glutamate residues and a pteridine ring bonded to PABA’s amino group. In plants, these molecules are produced in different parts of the cell. Pteridines are synthesized in the cytosol, whereas plastids produce PABA. These building blocks are then joined together inside mitochondria to give dihydropteroate (DHP), which is finally glutamylated to synthesize folates.

In nature, folates can have one-carbon units attached to their N5 and/or N10 positions at different oxidation levels. Some examples are the 5-formyl, 5-methyl, and 10-formyl derivatives. Alternatively, dihydrofolate (DHF) and other dihydro-derivatives may form by oxidation of the pteridine ring1. Folic acid per se is absent in nature.

Genetic engineering of folate biosynthesis

Pteridine overproduction Díaz de la Garza and colleagues initially targeted the first step in pteridine biosynthesis to engineer increased folate production. This reaction is catalyzed by guanosine triphosphate cyclohydrolase I (GCHI) (Fig. 1). Overexpression of this enzyme greatly increased the flux of pteridines in tomato fruit; however, the folate content was increased only 2 – 4 times. A limited supply of the other building blocks that constitute the folate molecule could be a limitation. Consistent with this idea was the observation that PABA pools in the engineered plants were very small4.

Figure 1. The tripartite folate biosynthesis pathway in plants. GTP, guanosine triphosphate. DHN, dihydroneopterin; -P, monophosphate; -PP, pyrophosphate -PPP, triphosphate; DHM, dihydromonapterin; HMDHP, hydroxymethyldihydropterin; all these pteridines are in purple. Aminodeoxychorismate (ADC) and p-aminobenzoate (PABA) are in blue. The red arrows represent the engineered GTP cyclohydrolase I (GCHI) and the ADC synthase (ADCS) enzymes.

Enhancement of p-aminobenzoate synthesis The limitation caused by small PABA pools in tomato was overcome by genetically engineering the first enzyme of PABA synthesis, aminodeoxychorismate synthase (ADCS) (Fig. 1). Tomatoes were transformed with the Arabidopsis ADCS coding sequence (AtADCS) (including its native chloroplast targeting peptide) under control of the fruit ripening-specific E8 promoter. RNA levels, measured at different ripening stages in the AtADCS+ transformants, were highest in fruit at the red stage. In these transformants, the level of accumulated PABA (56 nmol/g fresh weight) was 19-fold greater than in the empty vector controls. Although a marked increase in PABA was obtained in these transformants, folate production was the same in both controls and AtADCS+ tomatoes2. This result was consistent with the earlier observation that folate synthesis is also dependent on pteridine production4.

Folate biofortification To obtain tomato transformants with both PABA and pteridine overproduction, Diaz de la Garza and colleagues crossed hemizygous T0 AtADCS+ plants with one-insertion, hemizygous T1 GCHI+ plants. The development of plants and fruits was the same for double transgenic and nontransgenic segregants. Genomic DNA from the progenies of plants carrying both transgenes was screened by PCR. Control and transgenic tomato plants were harvested at the late red ripe stage and analyzed for pteridine and PABA content. The double transgenic fruit had 30-fold higher pteridine and PABA levels than controls and produced 25 times more folate (25 nmol/g fresh weight). No DHP, pteroate, or hydroxymethyldihydropterin pyrophosphate (HMDHP-PP) were found. These data suggest the presence of an additional restriction on the synthetic pathway2.

Other limitations on folate biofortification
Although the double transgenic tomatoes generated more folates, their pteridine and PABA pools were not depleted. On the other hand, DHP and DHF did not accumulate. These results indicate that there is a constraint in the folate production pathway at the level of DHP synthesis. Díaz de la Garza and colleagues (2007) think that the limitation lies either in the HMDHP- pyrophosphokinase step or in pteridine transport from cytosol to mitochondria.

Types of folate produced by the transformed tomatoes
5-methyl-THF was the main folate derivative found in double transgenics, pteridine overproducers, and wild type controls. DHF and folic acid were not detected. However, the monoglutamate form of 5-methyl-THF was the most abundant in GCHI+/AtADCS+ fruit, whereas 5-methyl-THF hexaglutamate was predominant in controls and fruit overexpressing GCHI+ alone2. Because a high concentration of glutamate can be readily found in red stage tomatoes, the low polyglutamylation of folate was not due to a limitation in this amino acid’s availability. Probably, folates were produced in double transgenic tomatoes at a rate that exceeds the capacity of folylpolyglutamate synthase. This was also the case in Lactococcus lactis genetically engineered to overproduce folate5. The total folate content in double transgenic tomatoes increased throughout ripening, whereas it didn’t change in the nontransgenic controls. This was expected because the expression of both transgenes was driven by the E8 promoter. Furthermore, in the double transgenic fruit, all types of folate derivatives increased, with polyglutamylation reaching a plateau at red stage followed by accumulation only of monoglutamyl folates7.

Safety concerns
As mentioned above, the double transgenic tomatoes had 30-fold higher PABA levels than controls. This amounts to <1 mg of PABA/100g portion. The U.S. Food and Drug Administration classifies the PABA food additive as GRAS (generally recognized as safe) when consumed up to 30 mg/day. Moreover, PABA has been proved in clinical trials to have low toxicities when consumed in quantities up to 12 g/day. Therefore, Díaz de la Garza and colleagues (2007) think it may be considered safe to eat biofortified tomatoes with the low levels of PABA observed.

The double transgenic fruit also contained higher pteridine levels—0.36 mg per portion. Unfortunately, little is known about pteridine in plant foods or its effect in humans. The pericarp of velvet bean (a medicinal legume) contains 470 nmol/g fresh weight of total pteridine, which is 25 times higher than the pteridine content of GCHI+/AtADCS+ tomatoes. On the other hand, it is well known that mammals catabolize pteridines and synthesize the pteridine tetrahydrobiopterin. Moreover, the total content of pteridines in the double transgenic tomato is <10% of the pteridines excreted/day in human urine. Based on these observations, Díaz de la Garza and colleagues suggest that pteridine levels in double transgenic tomatoes are likely to be harmless; however, they also state that further research is needed to establish the safety of plant foods containing this level of pteridines (2007).

Applications in gas ripened tomatoes and other plant foods
A general procedure in the food industry is to harvest mature green tomatoes and store them. Ripening is induced later by ethylene gas treatment before sending the fruit to the market. Hanson and Gregory’s laboratories applied this gas-ripening procedure to mature green double transgenic tomatoes until they reached the breaker stage. Fruits were allowed to ripen for 12 days. Tomatoes of the same double transgenic plants ripened on the vine were used as controls. The ethylene-ripened fruits contained 79% of the average folate levels of the controls. Also the types of folate and polyglutamylation levels in the gas ripened tomatoes were very similar to those in the controls2.

Is it feasible to apply the present strategy to fortify other plants with folates? Indeed, when an overexpression of GCHI was engineered in the small weed Arabidopsis, a three-fold increase in folates was obtained. Furthermore, when supplied with PABA, the transgenic plants accumulated 7 times more folate than the controls6. Thus, the technology for biofortification of tomatoes with folates has been well established in Hanson and Gregory’s laboratories, and the prospect of applying it to staple food plants such as tubers and cereal grains is very promising.

Conclusions
In the work reviewed, a marked increase in folate content in plants was demonstrated if both the pteridine and the PABA branches of the synthesis pathway were engineered. The experiments also advanced knowledge of the regulation of this pathway in plants.

The genetic engineering strategy employed was successful in the production of GCHI+/AtADCS+ transgenic tomato fruits producing 19-fold more folate than the controls. These folate levels are the highest thus far reported in plants. The double transgenic tomatoes contained 840 µg of folates/100 g fruit, which would provide the recommended dietary allowance for a pregnant woman in less than one standard serving. Furthermore, the size and appearance of the fruits did not change with the genetic transformation. Also, the folate content of double transgenic tomatoes was 7-fold higher than that of green leafy vegetables, which as mentioned before, are good folate sources in human diets.

References

1. Hanson AD, et al. (2001) One carbon metabolism in higher plants. Annu Rev Plant Physiol Plant Mol Biol 52, 119-137

2. Díaz de la Garza RI, et al. (2007) Folate biofortification of tomato fruit. Proc Natl Acad Sci USA 104, 4218-4222

3. Bailey LB, et al. (1999) Polymorphisms of methylenetetrahydrofolate reductase and other enzymes: metabolic significance, risks and impact on folate requirement. J Nutr 129, 919-922

4. Díaz de la Garza RI, et al. (2004) Folate biofortification in tomatoes by engineering the pteridine branch of folate synthesis. Proc Natl Acad Sci USA 101, 13720-13725

5. Sybesma W, et al. (2003) Increased production of folate by metabolic engineering of Lactococcus lactis. Appl Environ Microbiol 69, 3069-3076

6. Schubert KR, et al. (2005) Pteridines 16, 79

Claudia Lerma-Ortiz
Ph.D. Biochemistry
Consultant
Gainesville, Florida
tulum@bellsouth.net



SELECTED HIGHLIGHTS FROM 2007 BIO INTERNATIONAL CONVENTION
Tracy Sayler

The recent 2007 Biotechnology Industry Organization (BIO) International Convention held in Boston drew a record 22,366 attendees, a nearly 15 percent increase from the previous year. The Convention is the world’s largest event for the biotechnology industry. This year’s Convention drew representatives from 48 states and 64 countries, with one-third of attendees from outside the United States.

Keynote speakers were Queen Noor of Jordan and Michael J. Fox. Queen Noor discussed opportunities presented by biotechnology to address global health and poverty issues. Michael J. Fox, who founded the Michael J. Fox Foundation for Parkinson’s Research since being diagnosed with Parkinson’s disease in 1991, stressed the need for the biotechnology industry to continue to innovate and accelerate the translation of basic science into improved therapies for patients. The best drug for treating Parkinson’s has been around for 40 years, he pointed out. Fox likened treatment discovery to a high stakes poker game—it requires an investment, and sometimes we lose, sometimes we win, playing with the cards that are dealt. With biotechnology, the wild card may help win the game.

Agbiotech Goes Beyond Transgenics
Some people tend to think of biotechnology as only meaning "transgenic," when engineering transgenic traits into plants and animals is really just a small part of the biotech-driven revolution in agriculture. In reality, biotechnology also encompasses advances in genomics, bioinformatics, and molecular biology, and the bio industry needs to communicate the fact that more precise information about the genetic makeup of plants and animals (as well as plant and animal pests) can be used in non-transgenic ways to make better food, fuel, and fiber.

One panel of experts from the United States, New Zealand, and Australia described how information technology and agriculture are converging to create solutions in agriculture. "The convergence of information technology and molecular biology dramatically increases agriculture’s potential to supply fuel and animal feed as well as more nutritious food," said Sano Shimoda, president of California-based BioScience Securities.

Ray Riley, global head of corn and soybean product development for Syngenta, pointed out that agriculture based IT, such as gene sequencing and molecular markers technology, is increasingly becoming focused on consumer attributes "rather than just production by the pound." For example, source verification and DNA-based traceability is a cornerstone of food safety. And a better genetic understanding of cellulosic crops will help create more efficient biofuels.

Gerard Davis, CEO of Catapult Genetics (Australia/New Zealand), noted that global competition for grain drives research aimed at improving the efficiency of cattle and sheep in converting feed to meat, milk, and biofeedstuffs. He described how DNA analysis and genomics is supporting the targeted breeding of more efficient livestock.

Fruit breeding, too, is drawing on a suite of biotechnologies to create more novel, flavorful, and fragrant varieties with increased health promoting qualities. Gavin Ross, from New Zealand fruit developer HortResearch, pointed out that advances in germplasm IT, plant genomics, and flavor biotechnologies are generating tastier fruit varieties and new advances for the human health and flavor and fragrance industries, as well as ecologically better growing practices.

Opening Doors to Seed Banks
A key point made during the session titled 'Yours Mine or Ours; Who Owns the World’s Genetic Resources' was that the fear of big companies owning genetic seed resources is misaligned. In actuality, the problem is a hodge-podge of national policies that can inhibit access to plant genetic resources. Researchers and plant breeders frequently look to foreign sources of germplasm to find new traits for resistance to pest and diseases, yield improvement, and tolerance to environmental stresses. The emergence of new biotechnology-based plant breeding tools only heightens the importance of open access to germplasm, according to David Hegwood, USDA’s minister-counselor for agricultural affairs in Rome.

The International Treaty on Plant Genetic Resources for Food and Agriculture aims to improve international cooperation and the open exchange of crops and their genes that farmers all over the world have developed and exchanged over 10,000 years. Drafted in November 2001 after more than seven years of formal negotiations in the United Nations Food and Agriculture Organization, the ITPGRFA is the first effort to establish international rules governing access to genetic resources.

"It all starts with the seed," said Bernice Slutsky, Vice President for Scientific and International Affairs for the American Seed Trade Association, about access barriers to seed banks and germplasm vital to crop improvement. "Access, benefit sharing, innovation, and intellectual property protection all need to be factored together." ASTA advocates that the U.S. sign on to the treaty, details of which can be found online at http://www.fao.org/ag/cgrfa/itpgr.htm.

Cellulosic Ethanol: Closer Than You Might Think
As one daily newspaper pointed out, "three or four years ago, someone attending BIO’s annual convention could walk into one of the panel discussions on industrial biotechnology and practically hear crickets. It wasn’t exactly the hottest ticket in town." However, rising gas prices have changed that, and a number of well attended sessions at this year’s BIO focused on the booming biofuels market.

Industrial biotechnology is dedicated to achieving sustainable and renewable energy production. Use of specialized enzymes for generating biofuels can reduce or replace harsh chemicals that contaminate the environment and make the process more productive and efficient. In addition, new 'no cook' enzymes extract the sugars in corn at room temperature, greatly reducing energy inputs and improving the cost and environmental profile of ethanol made from corn starch. These advances in enzyme technology and microbial fermentation have increased the efficiency of corn ethanol production by 20 percent, from 2.5 gal/bu in 2000 to nearly 3 gal/bu today. Development of ethanol production from cellulosic biomass (such as corn stalks, wheat straw, or switchgrass) is also on the cusp of commercial production, due to dramatic advances in the development of cellulase enzymes. Industrial biotech companies have reduced the cost of the cellulose-digesting enzymes used to make ethanol by 30-fold since 2001, from over $5 per gallon of ethanol produced to under $0.20.

Brent Erickson, executive vice president in charge of the Industrial and Environmental Section at BIO, said that cellulosic ethanol is closer to commercialization than many think. In 2004, Iogen Corporation became the first company to begin commercial production of ethanol from cellulose, using biotech enzymes that convert wheat straw to clean burning ethanol. Other cellulosic ethanol biorefineries are under construction in York, Nebraska (Abengoa Bioenergy) and Emmetsburg, Iowa (Broin Companies). Biorefineries are also planned in Idaho (Iogen Corporation), New York (Mascoma Corporation), and Louisiana (Celunol/Diversa).

The 2008 BIO International Convention will be held June 17-20 in San Diego, California. Details will be available online at http://www.bio2008.org.

Tracy Sayler
Agricultural writer and ISB correspondent
Fargo, N.D.
tsayler@casselton.net




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