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Biotechnology Consultation
Note to the File
BNF No. 000087
Date: September 30, 2005
Subject: Biotechnology Notification File (BNF) 000087, maize transformation event LY038, OECD Unique Identifier-REN-ØØØ38-3
Keywords: Zea mays L., maize, corn, maize event LY038, dihydrodipicolinate synthase, Corynebacterium glutamicum, biolistic transformation, Cre-loxP recombination system
In a submission dated August 10, 2004, Monsanto Company (Monsanto) submitted to the Food and Drug Administration (FDA) a safety and nutritional assessment of genetically engineered lysine enhanced maize designated as maize event LY038. Monsanto provided additional information in support of this safety and nutritional assessment on November 1, 2004, and February 22, 2005. Monsanto concluded, based on the data and information summarized in their submission, that food and feed derived from maize event LY038 are as safe and nutritious as food and feed derived from currently available commercial varieties of maize.
Maize event LY038 was genetically engineered to increase the level of lysine in grain for animal feed applications. Grain from LY038 is intended for use in poultry and possibly swine diets. Poultry and swine diets based on maize (also known as corn) and soybean meal are typically supplemented with crystalline lysine to assure optimal animal growth. Monsanto expects the use of maize event LY038 as a feed ingredient to reduce or eliminate the need for lysine supplementation. Lysine is generally recognized as safe (GRAS) for use in animal diets.
Maize event LY038 contains the cordapA gene derived from Corynebacterium glutamicum. The gene encodes C. glutamicum dihydrodipicolinate synthase (cDHDPS). Dihydrodipicolinate synthase (DHDPS) is 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 is expected to result in the elevated levels of free lysine in the grain when compared to conventional maize.
Monsanto constructed an 8.8 kilobase (kb) Escherichia coli plasmid expression vector, PV-ZMPQ76. The plasmid contains three expression cassettes (cordapA, nptII, and amp). The cordapA cassette contains the cDHDPS gene, the nptII cassette contains the neomycin phosphotransferase type II gene from the E. coli transposon Tn5 (nptII), and the amp cassette contains the β-lactamase gene from a plasmid originally isolated from E. coli. The genetic elements associated with the respective open reading frames (ORFs) in each cassette are shown below in Table 1.
| Cassette Grouping | Genetic Elements | Description |
|---|---|---|
| cordapA | p-Glb I | The promoter from the Globulin 1 (Glb1) gene from Zea mays L. to drive cDHDPS expression. |
| rAct1 intron | Intron from the rice actin gene to enhance cDHDPS expression. | |
| maize DHDPS TP | The chloroplast targeting sequence for DHDPS from Z. mays L. to target cDHDPS to the chloroplast. | |
| cordapA | The coding region for cDHDPS from C. glutamicum in the lysine biosynthetic pathway, conferring resistance to lysine feedback inhibition. | |
| Glb1 3' UTR | The 3' nontranslated region from the Glb1 gene from Z. mays L. which directs the polyadenylation of the mRNA. | |
| nptII | loxP | Recombination site recognized by Cre recombinase. |
| CaMV 35S promoter | Cauliflower mosaic virus promoter to drive nptII expression. | |
| nptII | The coding region for nptII from Tn5, a transposon isolated from E. coli, confers the paromomycin resistance that permits the selection of cells containing the expression cassette. | |
| Ble | A 0.153 kb portion of the 0.378 kb bleomycin gene from Tn5. | |
| NOS 3' | The 3' nontranslated region of the nopaline synthase (NOS) coding sequence from Agrobacterium tumefaciens which directs the polyadenylation of the mRNA. | |
| loxP | Recombination site recognized by Cre recombinase. | |
| amp | Bacterial promoter and coding sequence for β-lactamase | Confers ampicillin resistance that permits the selection of cells containing the expression cassette in E. coli. |
Following amplification in E. coli, the plasmid, PV-ZMPQ76, was digested with Xho I and a 5.9 kb fragment containing the cordapA and npt II gene cassettes was isolated. Maize callus material from maize inbred line H99 was transformed with this 5.9 kb Xho I fragment by a particle acceleration methodology (biolistic method). Plants were regenerated from the callus tissue cells, grown in the presence of paromomycin, and assayed for the presence of the cordapA gene using standard PCR methodology. Plants that were identified as expressing the cordapA gene were further propagated.
The regenerated plants containing the cordapA gene were crossed with maize plants engineered to express 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 eventually segregated away from the cordapA gene through subsequent breeding. The removal of the npt II and cre genes was verified by event-specific PCR analyses and by Southern blot analyses (also known as genomic DNA gel blot analysis) conducted during the LY038 development process.
Monsanto used restriction enzyme digestion and Southern blot analyses to support their conclusion that LY038 contained one intact copy of the cordapA gene cassette, which was inserted into the maize genome at a single site. Monsanto also confirmed that there were no additional elements from PV-ZMPQ76, linked or unlinked to the intact gene cassette, in the genome of LY038. Furthermore, Monsanto confirmed that LY038 did not contain either intact or partial DNA fragments from the npt II gene cassette or cre gene cassette or any detectable backbone sequences from the plasmid vector PV-ZM003.1 PCR analyses were used to confirm that the organization of the genetic elements of the DNA fragment inserted into LY038 is identical to that of the cordapA gene cassette in plasmid PV-ZMPQ76.
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 (χ2) values. Monsanto reported that these segregation data indicate 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.
Dihydrodipicolinate synthase (DHDPS) is a member of the lyase subfamily of pyruvate-dependent class I aldolases found in a wide range of organisms including bacteria, rodents and humans. DHDPS mediates a critical rate-limiting step in the lysine biosynthetic pathway that in maize is controlled by lysine feedback inhibition. The enzyme catalyzes the condensation of L-aspartate-4-semialdehyde and pyruvate to form 2, 3-dihydrodipicolinate, which is converted to lysine through a series of subsequent enzymatic reactions. The cordapA gene expressed in maize LY038 encodes dihydrodipicolinate synthase from Corynebacterium glutamicum (cDHDPS), a non-pathogenic species of coryneform bacteria that are widely distributed in nature. C. glutamicum is used in the commercial production of several amino acids including lysine. cDHDPS is less sensitive to lysine feedback inhibition than the native maize DHDPS. This results in the accumulation of free lysine in the grain of LY038 at levels above those typically found in conventional maize.
Monsanto compared the amino acid sequences of cDHDPS to the amino acid sequences of DHDPS proteins from other bacteria and plants. Monsanto reports that the sequence identity and similarity of cDHDPS to other DHDPS isoforms ranged from 27 to 37 percent and from 36 to 47 percent, respectively. The sequence of the native maize enzyme is 29.4% identical and 38.5% similar to the sequence of cDHDPS.
Monsanto produced adequate quantities of cDHDPS in E. coli2 to be able to conduct studies on potential allergenicity and safety of cDHDPS. Monsanto compared the physicochemical and functional characteristics of the LY038-derived cDHDPS and the E. coli-derived cDHDPS proteins using the following methods:
Monsanto provided the results of the above experiments and concluded the cDHDPS proteins derived from E. coli and LY038 were equivalent.
Monsanto evaluated the levels of cDHDPS protein in LY038 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. Monsanto used the negative segregant LY038(-) as the negative control and the 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. According to Monsanto, these results indicate that cDHDPS is expressed primarily in the grain tissue. Monsanto estimated the potential maximum daily intake of the cDHDPS protein for broilers, young pigs, and finishing pigs to be 1482, 962, 598 μg/kg BW/day, respectively.3
Monsanto states that the potential allergenicity of cDHDPS protein was assessed by searching for amino acid sequence similarity between cDHDPS and known allergen protein sequences in the allergen database AD4 using FASTA sequence alignment tool and by evaluation of the stability of cDHDPS protein using an in vitro gastric digestion method.
The amino acid sequence alignment analysis revealed 23.9% identity between cDHDPS and Mercurialis annua profilin allergen over a 92 amino acid window. Monsanto noted that the length of the overlap is relatively short when compared to the full length (303 amino acid) cDHDPS protein and that the longest stretch of the contiguous amino acid identities consisted of three amino acids. Monsanto concluded that, based on this result, cross-reactivity between cDHDPS and M. annua profilin allergen is highly unlikely. Monsanto also stated that no other immunologically relevant sequences, such as eight contiguous amino acid identities, were detected when cDHDPS amino acid sequence was compared to the allergen sequences in AD4 database and concluded that cDHDPS protein is unlikely to share structurally and immunologically relevant sequence similarities with known allergens.
Monsanto provided data on the in vitro digestibility of the cDHDPS protein in simulated gastric fluid containing pepsin. Monsanto reported that greater than 96% of the cDHDPS protein was digested in SGF within 30 seconds when analyzed using Colloidal Brilliant Blue G-stained polyacrylamide gels and greater than 98% of the protein was digested within 30 seconds when the digest was analyzed using Western blot analysis. Monsanto concluded that these results support the conclusion that cDHDPS has a low allergenic potential.
Monsanto conducted an acute high dose oral toxicity study in mice using the E. coli-derived cDHDPS protein. Monsanto stated that a single gavage dose of 800 mg of cDHDPS/kg body weight did not result in any mortalities or adverse clinical reactions in mice.
The grain of LY038 maize contains elevated levels of lysine4 and its catabolites saccharopine and α-aminoadipic acid. To address the safety of lysine catabolites, Monsanto noted that animals are continually exposed to saccharopine and α-aminoadipic acid in the normal course of endogenous lysine metabolism. These compounds are degraded and they ultimately become substrates for the tricarboxylic acid cycle, entering as acetoacetyl-CoA. Monsanto concluded that, even if farm animals consumed large amounts of LY038 maize grain, that 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. Monsanto cited literature articles reporting studies in pigs, rats, and broilers as evidence that there were no adverse effects when animals were exposed to saccharopine or α-aminoadipic acid. Furthermore, Monsanto stated that, based on published literature and analyses performed at their own facility, substantial amounts of α-aminoadipic acid and saccharopine are found in foods commonly consumed by people. For example, α-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.
Maize, Zea mays L., originated in Mexico and was grown as a food crop as early as 2700 B.C. Maize is a highly productive crop, currently yielding an average of 142 bushels per acre in the U.S. (National Corn Growers Association, 2004). Most of the annual maize production is fed to cattle, chickens, and swine either as intact or processed grain, or as dry or wet milling byproducts. Maize forage is fed to ruminants. Maize is also used in human food as whole kernel grain or processed into maize-based food ingredients such as starch, high fructose corn syrup, and corn oil.
Monsanto evaluated the composition of forage and grain from maize LY038 (test material) using forage and grain from the negative segregant LY038(-) as the control material. The LY038(-) segregant has a genetic background representative of LY038, but does not contain the cordapA gene. 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.
Monsanto performed statistical analyses of the compositional data using a mixed model analysis of variance method. The levels of individual components of forage and grain from LY038 and LY038(-) were compared. Analytical data were provided for five individual test sites and for the "combined site." The data for the "combined site" were obtained by aggregating the analytical data from five individual test sites. Statistically significant differences were determined at the 5% significance level (P≤0.05). Using analytical data obtained for each of the 20 conventional hybrid lines, a "99% tolerance interval" was calculated to contain, with 95% confidence, 99% of the values obtained for the conventional corn hybrids. Historical compositional data, based on studies previously conducted by Monsanto, as well as published literature values were also provided to assess whether statistically significant differences in the composition of the test and control maize are biologically meaningful.
Forage was harvested at the early dent stage (R6 growth stage). Compositional analyses of the forage samples included:
Monsanto reported that for the "combined site" there were no statistically significant differences in the mean levels of all measured components except for phosphorus. The level of phosphorus was lower (P ≤ 0.05) in LY038 than in LY038(-).5 The levels of all components including phosphorus fell within literature ranges and within the 99% tolerance interval calculated for the conventional maize.
Grain samples were harvested at maturity (R6 growth stage) and analyzed for 75 components. Analytical data for the following components were provided:6
Monsanto reported 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 the field maize (OECD, 2002). As expected, the levels of lysine catabolites, saccharopine, and α-aminoadipic acid were also elevated in the LY038 grain.
Monsanto provided statistical evaluation of data obtained for the individual test sites5 and for the "combined site." For the "combined site," Monsanto reported statistically significant differences between LY038 and LY038(-) as detailed in the following table:
| Levels Higher in LY038 than LY038(-) | Levels Lower in LY038 than LY038(-) |
|---|---|
| Crude protein | Total fat |
| Fatty acids: oleic acid, linolenic acid, arachidic acid | Fatty acids: linoleic acid, eicosenoic acid |
| Minerals: zinc, copper | Minerals: calcium, manganese |
| Fiber: TDF, NDF | Vitamin E |
| Amino acids (when calculated as percent dry weight): alanine, aspartic acid, cysteine, glycine, leucine, methionine, phenylalanine, serine, valine, lysine, free lysine Amino acids (when calculated as percent of total amino acids): lysine |
Amino acids (when calculated as percent of total amino acids): glutamic acid, histidine, isoleucine, and phenylalanine |
| Folic acid | |
| Saccharopine | |
| L-pipecolic acid |
Monsanto noted that some of the statistically significant differences in the composition between LY038 and LY038(-) reflect the intended increase in the levels of lysine in LY038. Monsanto evaluated the remaining compositional differences and reported that the levels of LY038 components associated with statistically significant differences were either within the 99 % tolerance level and/or within historical ranges for maize based on Monsanto's studies or ranges reported in the literature. Monsanto considered these compositional differences to be biologically irrelevant.
Monsanto reported that 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).
Monsanto concluded that with the exception of total and free lysine and the lysine catabolites saccharopine and α-aminoadipic acid, the LY038 grain is compositionally equivalent to the conventional maize grain. As discussed earlier in section 5, Monsanto presented arguments based on published studies that even though the levels of lysine catabolites are increased the LY038 grain, the grain is safe for use in animal feed and human food.
Monsanto noted that typically maize-based diets for poultry and swine are deficient in the amino acid lysine and that 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 wholesomeness of LY038 when used as animal feed, Monsanto conducted a 42 day feeding study on growing broiler chickens, the primary intended market for LY038.
In the 42-day feeding study, bird performance was compared when fed LY038 versus the control, LY038(-), and 4 conventional maize varieties with and without added crystalline lysine, making a total of 11 treatments. Diets were formulated to contain the maximum possible amount of maize (approximately 64% and 71% of the starter and grower/finisher diets, respectively). The treatment diets without added lysine, LY038(-) and conventional maize diets, were formulated to a lysine level below that required for optimal growth with the remainder of the essential amino acids meeting or exceeding the requirements for optimal growth. Treatment diets with added lysine were formulated to a lysine level equivalent to the lysine level in the LY038 treatment with the remainder of the essential amino acids meeting or exceeding the requirements for optimal growth.7
The broilers consumed approximately 82 g of grain/kg body weight per day when averaged over the 42-day feeding trial which is in line with industry expectations. Monsanto reported a statistically significant increase in broiler growth rate and feed efficiency in broilers receiving LY038(-) and conventional maize diets supplemented with lysine versus the same diets without added lysine. In addition, they reported no statistically significant differences in growth rate or feed efficiency in broilers fed LY038 diet versus birds fed LY038(-) or the four conventional varieties supplemented with crystalline lysine. Monsanto reported a statistically significant increase in growth rate and feed efficiency in broilers fed LY038 versus birds fed LY038(-) and the four conventional varieties not supplemented with lysine. Chick mortality was low and was random without any relationship to treatment. No unexpected effects on bird health were observed with the feeding of LY038 grain.
Monsanto has concluded that maize event LY038 is not materially different in composition, safety, or any other relevant parameter from maize now grown, marketed, and consumed in the U.S, with the exception of the intentionally-increased lysine content in the grain. At this time, based on Monsanto's data and information, the agency does not have any further questions and considers Monsanto's consultation on maize event LY038 to be complete.
W. D. Price, Ph.D., PAS
(1)As described in Monsanto's submission, plasmid PV-ZM003 was used in the transformation of maize to generate plants expressing Cre recombinase.
(2)The cDHDPS protein produced in E. coli was encoded by the cordapA gene with, at the N-terminus, three additional amino acids from the C-terminus of the maize dihydrodipicolinate transit peptide sequence.
(3)Monsanto stated that they intend to segregate LY038 from conventional maize. Nevertheless, they estimated the potential human exposure to the cDHDPS protein assuming various degrees of inadvertent commingling of the LY038 grain with the conventional maize grain. Monsanto's studies indicate that the cDHDPS protein does not possess characteristics of allergens and toxins and is easily digestible. The cDHDPS protein is an enzyme that is functionally equivalent and structurally closely related to other DHDPS enzymes that commonly occur in food (for example in corn, soy, wheat, rice, or spinach) and are safely consumed. Thus, the presence of the cDHDPS protein in LY038 does not raise food safety issues in the event of human consumption of LY038 maize at levels typically expected for conventional maize.
(4)FDA notes that while the level of lysine in LY038 is elevated, it is within the literature range reported for maize varieties consumed by humans. Therefore, consumption of LY038 would not change the overall dietary lysine intake of humans.
(5)Monsanto observed several differences in the level of nutrients within the locations; these were not consistently altered across locations.
(6)Grain samples were analyzed for a total of 75 components. The levels of 18 components (sodium, furfural, cadaverine, 2, 6-diaminopimelic acid, and 14 fatty acids) were below the limits of quantification (LOQ) of the respective assays in all of the grain samples. Therefore, data for these components were not statistically analyzed and were not provided in the submission. In addition, the levels of α-aminoadipic acid were below the LOQ in the majority of LY038(-) and conventional maize reference samples. Consequently, the levels of α-aminoadipic acid in LY038 grain were provided but were not subjected to the statistical analysis.
(7)Monsanto assayed the 11 treatment diets for amino acids, but the results of the assays were variable, and in several cases did not agree with the targeted formulated values. A PCR analysis was conducted on each corn variety prior to mixing of the diet to determine pureness, and all were as expected with the exception of LY038. For LY038, the PCR showed > 99% LY038 trait as expected, but also showed up to 20.5% MON 810 (Cry1Ab; pesticide resistant). MON 810 was the subject of FDA Biotech Notification File 34 and a successful consultation was completed. Therefore, Monsanto considered MON 810 as an inert ingredient that did not impact the objectives and interpretation of the study.
