GM Crop Database

Database Product Description

LY038 (REN-ØØØ38-3)
Host Organism
Zea mays (Maize)
Trait
Enhanced lysine level.
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use

Production for livestock feed.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2007
Canada 2006 2006 2006
Colombia 2009
Japan 2007 2007 2007
Mexico 2007
Philippines 2006 2006
Taiwan 2006
United States 2005 2005 2006

Introduction Expand

LY038 maize has been genetically modified to express the cordapA gene from Corynebacterium glutamicum. This gene encodes for a lysine –insensitive dihydropicolinate synthase (cDHDPS) enzyme, a regulatory enzyme in the lysine biosynthetic pathway. The activity of the native maize DHDPS is regulated by lysine feedback inhibition. Since the cDHDPS enzyme is less sensitive to lysine feedback inhibition, its expression in maize LY038 results in elevated levels of free lysine in the grain when compared to conventional maize. The expression of the cordapA gene is under the control of the maize Glb1 promoter, which directs cDHDPS expression predominantly in the germ of the seed, resulting in accumulation of lysine in the grain.

Maize-soybean meal based diets formulated for poultry and swine are characteristically deficient in lysine and require the addition of supplemental lysine for optimal growth and production of these animals. The development of LY038 maize provides an alternative to direct supplementation of poultry and swine diets by increasing the amount of lysine in the maize portion of the feed.

There are no selectable markers present in the final LY038 event as these were removed after the selection of the initial transgenic event using a site-specific recombination system. The recombination-mediated removal of the selectable marker was accomplished by crossing with a second transgenic maize plant expressing a specific recombinase gene, and subsequent crossing and selection resulted in the isolation of lines containing only the cordapA gene as inserted DNA.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
cordapA dihydrodipicolinate synthase AA promoter from globulin 1 (Glb1) gene from Zea mays rice actin gene intron (rAct1) and Z. mays chloroplast transit peptide sequence for DHDPS Glb1 gene 3' non-translated region from Z. mays 1 functional

Characteristics of Zea mays (Maize) Expand

Center of Origin Reproduction Toxins Allergenicity

Mesoamerican region, now Mexico and Central America

Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.

No endogenous toxins or significant levels of antinutritional factors.

Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Modification Method Expand

LY038 maize was developed using microprojectile bombardment with DNA-coated gold microprojectiles accelerated into callus derived from maize inbred line H99. The DNA used comprised a 5.9 Kb fragment of plasmid PV-ZMPQ76 containing the cordapA cassette and an nptII cassette as a selectable marker which was flanked by loxP recombination sites. Selection of transgenic callus and regenerated plants was performed by growth on paromomycin and plants positive for the cordapA gene by PCR were crossed to another maize line engineered to express the Cre recombinase protein. The Cre recombinase in the resulting hybrid initiated the excision of the DNA fragment, containing the nptII gene cassette flanked by the loxP sites, and splicing together of the two flanking chromosomal DNA fragments. The progeny were screened for plants that did not contain the nptII gene cassette, but contained the cordapA gene cassette. The cre gene, which was also integrated into the maize genome, was segregated away from the cordapA gene through subsequent breeding.

Characteristics of the Modification Expand

The Introduced DNA:

Plasmid PV-ZMPQ76 contained three expression cassettes – cordapA, nptII, and amp which was used for selection in E.coli. The 5.9 Kb fragment of this plasmid used for transformation contained only the cordapA and nptII cassettes and in the final product only the cordapA cassette was present. This was confirmed by event-specific PCR analyses and Southern blot analyses, which also confirmed that LY038 contained one intact copy of the cordapA gene cassette. No additional elements from PV-ZMPQ76 were identified in the genome and there were no intact or partial DNA fragments from PV-ZM003 (used to transform lines expressing the Cre recombinase).

Genetic Stability of the Trait:

Heritability and stability of the cordapA gene cassette in LY038 were determined on plants from: 1) the F1' generation (prior to excision of the npt II marker gene); 2) the F3 generation (after the excision of the npt II marker gene); and 3) the F4 generation (after two rounds of backcrossing to conventional inbred lines). The expected segregation ratios were 1:1, 3:1, and 3:1, for the F1', F3, and F4 generations, respectively. There were no significant differences in the observed-to-expected segregation ratios for the LY038 cordapA gene cassette over five plant generations, as demonstrated by the chi-square values. These segregation data indicates a single-locus Mendelian inheritance pattern for the transgene. These data are also consistent with the molecular analyses which suggested that the cordapA transgene was stably integrated at a single site in the genome. Southern blot analysis of LY038 genomic DNA established that the inserted DNA was stably transferred across seven generations and that cre and nptII cassette elements were absent in LY038.

Environmental Safety Considerations Expand

Field Testing:

Numerous field trials were conducted in 2002 and 2003 in a variety of locations to evaluate LY038 corn. In each of these trials, LY038 was compared to a negative segregant (a sister line in which the cordapA transgene was not present) referred to as LY038(-) as well as several control and reference hybrids. Standard field trials included an evaluation of dormancy and germination, ecological evaluations (plant interactions with insect pests, disease, and abiotic stresses), phenotypic evaluations, and compositional changes. Data addressing the above categories were collected in order to assess possible effects from introduction of the cordapA gene and its associated regulatory sequences.

Dormancy and germination testing revealed no differences found between LY038 corn and the reference hybrids. There were slight qualitative differences between LY038 corn and the controls identified in the ecological evaluations, but all of the measurements were within the range of incidence observed for the reference hybrids. For the phenotypic evaluations, there were 14 phenotypic characteristics evaluated during field testing. While there were some small qualitative statistical differences found, such as decreased seedling vigor and a small increase in plant height; these varied between years and were all within the ranges observed for the reference corn hybrids. Analysis of phenotypic characteristics data showed no significant biological differences between the reference corn control populations and LY038 corn, or differences outside the range of conventional corn norms.

The only unusual observation was the appearance of a white-leaf phenotype in LY038 corn in some field test locations. The white-leaf phenotype occurs at germination and persists only to the V2 stage (when the second collar appears on the second leaf). A similar white-leaf or decreased chlorophyll content phenotype has been observed in other plant species that accumulate high concentrations of lysine. In some cases this trait is associated with a loss of apical dominance and other growth abnormalities, however no such characteristics were observed in the LY038 plants in these field tests.

Outcrossing:

Since pollen production and viability were unchanged by the genetic modification resulting in LY038, pollen dispersal by wind and outcropping frequency should be no different than for other maize varieties. Gene exchange between LY038 maize and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In the United States, where there are no plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Feral species in the United States related to corn cannot be pollinated due to differences in chromosome number, phenology (periodicity or timing of events within an organism’s life cycle as related to climate, e.g., flowering time) and habitat.

Maize (Zea mays ssp. mays) freely hybridizes with annual teosinte (Zea mays ssp. mexicana) when in close proximity. These wild maize relatives are native to Central America and are not present in the United States, except for special plantings. Tripsacum, another genus related to Zea, contains sixteen species, of which twelve are native to Mexico and Guatemala. Three species of Tripsacum have been reported in the continental United States: T. dactyloides, T. floridanum and T. lanceolatum. Of these, T. dactyloides, Eastern Gama Grass, is the only species of widespread occurrence and of any agricultural importance. It is commonly grown as a forage grass and has been the subject of some agronomic improvement (i.e., selection and classical breeding). T. floridanum is known from southern Florida and T. lanceolatum is present in the Mule Mountains of Arizona and possibly southern New Mexico. Even though some Tripsacum species occur in areas where maize is cultivated, gene introgression from maize under natural conditions is highly unlikely, if not impossible. Hybrids of Tripsacum species with Zea mays are difficult to obtain outside of the controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduced fertility.

Weediness Potential:

No competitive advantage was conferred to LY038 that would render maize weedy or invasive of natural habitats, since none of the reproductive or growth characteristics were modified. Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Zea mays is not invasive and is a weak competitor with very limited seed dispersal. Volunteers can occur in fields, the year following cultivation, when maize is grown in rotation with other crops. Although maize volunteers are a minor weed problem, these can cause harvesting problems.

Impact on Biodiversity:

LY038 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in the United States is remote, it was determined that the risk of transferring genetic traits from LY038 maize to species in unmanaged environments was insignificant.

Food and/or Feed Safety Considerations Expand

Toxicity and Allergenicity:

Amino acid sequence alignment analysis, using the FASTA algorithm, revealed 23.9% identity between cDHDPS and Mercurialis annua profilin allergen over a 92 amino acid window. However, the length of the overlap is relatively short when compared to the full length (303 amino acid) cDHDPS protein and the longest stretch of the contiguous amino acid identities consisted of three amino acids. Based on this result, it is considered that cross-reactivity between cDHDPS and M. annua profilin allergen is highly unlikely. No other immunologically relevant sequences were detected when cDHDPS amino acid sequence was compared to the allergen sequences in AD4 database and it was concluded that cDHDPS protein is unlikely to share structurally and immunologically relevant sequence similarities with known allergens.

In in vitro digestion studies, greater than 96% of the cDHDPS protein was digested in simulated gastric fluid within 30 seconds supporting the conclusion that cDHDPS has a low allergenic potential.

An acute high dose oral toxicity study in mice using E. coli-derived cDHDPS protein involving a single gavage dose of 800 mg of cDHDPS/kg body weight did not result in any mortalities or adverse clinical reactions in mice.

Dietary Exposure:

The levels of cDHDPS protein in LY038 were evaluated in tissue samples collected from plants grown at five field sites in the U.S., using an ELISA assay with goat polyclonal antibody specific for cDHDPS. The negative segregant LY038(-) was used as the negative control and E. coli-derived cDHDPS as the reference standard. The mean cDHDPS protein levels in LY038 grain, forage in the early dent stage, whole plant, forage root, root, and pollen tissues were 26.0, 0.94, 0.081, 0.069, 1.5, and 0.78 µg/g dry weight, respectively. cDHDPS in leaf tissue was measured at four time points throughout the growing season and was not detected at the assay detection limit of 0.013 µg/g fresh weight. These results indicate that cDHDPS is expressed primarily in the grain tissue as expected from the use of the Glb1 promoter. The estimated potential maximum daily intake of the cDHDPS protein for broilers, young pigs, and finishing pigs was calculated to be 1482, 962, 598 µg/kg BW/day, respectively.

Humans consume relatively little whole kernel or processed maize, compared to maize-based food ingredients. Maize is a raw material for the manufacture of starch, the majority of which is converted to a variety of sweetener and fermentation products, including high fructose syrup and ethanol. Maize oil is commercially processed from the germ. As the target use of LY038 is for animal feed, dietary exposure to humans of the cDHDPS protein will be significantly lower than the estimated levels calculated above for animals fed with this maize.

Lysine Catabolites:

The grain of LY038 maize contains elevated levels of lysine and its catabolites saccharopine and ?-aminoadipic acid. Animals are continually exposed to saccharopine and ?-aminoadipic acid in the normal course of endogenous lysine metabolism and these compounds are degraded and ultimately become substrates for the tricarboxylic acid cycle, entering as acetoacetyl-CoA. Even if farm animals consumed large amounts of LY038 maize grain, the saccharopine would be completely degraded because of the excess capacity of saccharopine dehydrogenase in their liver and it would neither cause a safety problem to animals, nor would it accumulate in the tissue to pose a problem to humans consuming meat. Articles in the literature report studies in pigs, rats, and broiler chickens showing no adverse effects when animals were exposed to saccharopine or ?-aminoadipic acid. Furthermore, substantial amounts of ?-aminoadipic acid and saccharopine are found in foods commonly consumed by people. For example, ?-aminoadipic acid is found in lentils, garden peas, broccoli, cauliflower, green beans and lettuce and saccharopine is normally found in asparagus, edible mushrooms, lettuce, lentils and brie cheese.

Nutritional and Compositional Data:

The composition of forage and grain from maize LY038 was evaluated using forage and grain from a negative segregant LY038(-), which did not contain the cordapA gene, as the control material. LY038 and LY038(-) maize lines were grown during the 2002 field season at five replicated fields in a randomized complete block design with three replicates per block. In addition, 20 conventional maize hybrids (reference material) were grown, four per site, to determine the amount of variation in nutrient composition that might be expected at each site. Forage and grain samples were collected from all plots and analyzed for nutritional components, anti-nutrients and secondary metabolites. In addition, six lysine-related secondary metabolites from lysine biosynthetic and catabolic pathways, as well as free (not incorporated into protein) and total lysine, were analyzed in LY038, LY038(-), and the conventional maize hybrid grain samples.

Forage was harvested at the early dent stage. Compositional analyses of the forage revealed no statistically significant differences in the mean levels of all measured components except for phosphorus. The level of phosphorus was lower in LY038 than in LY038(-). However, the levels of all components, including phosphorus, fell within ranges detailed in the literature and within the 99% tolerance interval calculated for the conventional maize hybrids grown as controls.

Grain samples were harvested at maturity (R6 growth stage) and analyzed for 75 components. Analytical data showed that, as intended, the levels of free and total lysine were higher in the LY038 grain than in the control LY038 (-) grain. However, the range of total lysine levels in LY038 was within the range reported for field maize. As expected, the levels of lysine catabolites, (saccharopine and ?-aminoadipic acid) were also elevated in the LY038 grain.

Other statistically significant compositional differences were either within the 99 % tolerance level and/or within historical ranges for maize based on the field studies or ranges reported in the literature. No statistically significant differences were observed between LY038 and LY038(-) in the levels of antinutrients (phytic acid and raffinose) and secondary metabolites (ferulic acid and p-coumaric acid).

Bioefficacy and Safety of LY038 in Broiler Chickens:

Maize-based diets for poultry and swine are typically deficient in the amino acid lysine and these diets are commonly supplemented with crystalline lysine to meet the animal's amino acid requirements. LY038 was developed to provide maize grain with consistently higher lysine content. To assess the nutritional value of the increased level of lysine in LY038, and the wholesomeness of LY038 when used as animal feed, a 42 day feeding study was conducted on growing broiler chickens, the primary intended market for LY038. A statistically significant increase in growth rate and feed efficiency was observed in broilers fed LY038 versus birds fed LY038(-) or four conventional varieties not supplemented with lysine. Chick mortality was low and random without any relationship to treatment. No unexpected effects on bird health were observed with the feeding of LY038 grain.

Abstract Collapse

Maize, or corn (Zea mays L.) is grown commercially in over 100 countries with a combined harvest of nearly 700 million metric tonnes in 2006. The top five producers of maize in 2005 were the United States, China, Brazil, Argentina, and Mexico, accounting for 70% of world production. Maize is grown primarily for its kernel (grain), the majority of which is used for animal feed, but with significant amounts refined into products used in a wide range of food, medical, and industrial goods.

Maize is a raw material for the manufacture of starch, the majority of which is converted by a complex refining process into sweeteners, syrups, and fermentation products, including ethanol. Maize oil is extracted from the germ of the maize kernel. Only a small proportion of the whole kernel is consumed by humans (e.g., corn meal, grits, oil), while refined maize products such as sweeteners, starch, and oil are abundant in processed foods (e.g., breakfast cereals, dairy goods, chewing gum). Maize is also processed into masa, which is used for tortillas, tacos and corn chips.

In the United States maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, however a growing amount is now being used for the production of ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, car parts. Refined maize products are also used in bioproducts such as antibiotics.

The transgenic maize line LY038 was genetically engineered to increase the level of the amino acid lysine in the grain for animal feed, primarily for poultry and swine. Poultry and swine diets based on maize grain are usually supplemented with lysine. The use of LY038 as a feed ingredient is expected to reduce or eliminate the need for lysine supplementation. The maize line LY038 contains the cordapA gene from Corynebacterium glutamicum , which was introduced using micro-projectile bombardment of maize callus cells.

The cordapA gene isolated from Corynebacterium glutamicum, codes for the enzyme dihydrodipicolinate syntase (cDHDPS). This enzyme is active in the lysine metabolic pathway and mediates a critical rate-limiting step, that in maize, is controlled by feedback inhibition. DHDPS catalyzes the condensation of L-aspartate-4-semialdehyde and pyruvate , resultling in the synthesis of 2,3-dihydrodipicolinate, a substrate in the lysine metabolic pathway. The DHDPS encoded by cordapA is less sensitive to feedback inhibition than the native maize DHDPS. The reduction in feedback inhibition results in higher levels of free lysine accumulating in the grain of LY038, at levels higher than those typically found in conventional maize.

Links to Further Information Expand

Canadian Food Inspection Agency Food Standards Australia New Zealand Health Canada Novel Foods Japanese Biosafety Clearing House, Ministry of Environment Philippines Department of Agriculture, Bureau of Plant Industry U.S.Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Friday, March 26, 2010