Database Product Description

3272 (SYN-E3272-5)

Host Organism

Zea mays (Maize)

Trait

Modified amylase for ethanol production

Trait Introduction

Agrobacterium tumefaciens-mediated plant transformation.

Proposed Use

Production of maize grain primarily for industrial ethanol.

Product Developer

Syngenta Seeds, Inc.

Summary of Regulatory Approvals

Introduction Expand

Maize (Zea mays L), otherwise known as corn, is the world’s third leading cereal crop, behind wheat and rice, and is grown in over 25 countries worldwide. The majority of grain and forage derived from maize is used as animal feed, however maize also has a long history of safe use as food for human consumption. The grain can be processed into industrial products such as ethanol (by fermentation), and highly refined starch (by wetmilling) and sweetener products. In addition to milling, the maize germ can be processed to obtain corn oil and numerous other products.
The majority of maize is grown for animal feed production and human food, however an increasing percentage is being used for the production of ethanol as a biofuel alternative to petroleum products from fossil sources. The hydrolysis of the large starch molecules in maize kernels is the first step in ethanol production from maize and the themostable alpha-amylase in Event 3272 is intended to increase the efficiency of this process by operating at high temperatures.

Maize line 3272 has been genetically modified to express a thermostable alpha-amylase enzyme (AMY797E) for use in dry-grind fuel ethanol production in the United States. Amylase is used to hydrolyse starch into smaller sugar subunits, which is the first step in producing ethanol from plants. Amylases used for ethanol production need to be able to work at high temperatures and low calcium concentrations. Plants such as maize naturally contain amylases, however these are destroyed when corn is subjected to the high processing temperatures necessary for ethanol production, making it necessary to add microbially-produced amylase preparations. The use of Event 3272, expressing a highly thermostable amylase, bypasses this step.

Summary of Introduced Genetic Elements Expand

Code	Name	Type	Promoter, other	Terminator	Copies	Form
pmi	mannose-6-phosphate isomerase	SM	ZmUbiInt (Zea mays poly-ubiquitin gene promoter and first intron)	A. tumefaciens nopaline synthase (nos) 3'-untranslated region	1
amy797E	thermostable alpha-amylase	PQ	GZein promoter - from maize 27-kDa zein gene PEPC9 - intron 9 from maize phospho-enol-pyruvate carboxylase gene	35S terminator from cauliflower mosaic virus	1	Chimeric alpha-amylase

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

Maize line 3272 was produced by Agrobacterium-mediated transformation of immature embryos derived from the proprietary line A188 of Zea mays (maize) using the transformation vector pNOV7013. The T-DNA segment of this plasmid contained, between the right and left borders, the amy797E and pmi genes and their regulatory elements.

The chimeric amy797E gene was generated to combine the best features of three thermostable amylase enzymes isolated from Thermococcus species bacteria, designated as BD5031, BD5064 and BD5063 (Richardson et al., 2002). Fragments from the three parental genes were combined (in the same relative position) based on the natural sequence homology to create a library of recombinant alpha-amylase genes. The chimeric AMY797E aplha-amylase enzyme (alternatively known as “797GL3”) was identified by screening these recombined enzymes and is composed of four fragments from BD5031, two fragments from BD5064 and three fragments from BD5063. The amy797E gene includes fusions of a 19 amino acid N-terminal maize gamma-zein signal sequence and a C-terminal endoplasmic reticulum retention signal (Lanahan et al., 2003). The maize gamma-zein signal sequence and the ER retention signal provide for protein targeting to and retention in the endoplasmic reticulum of the cell, respectively. The alpha-amylase coding region of the amy797E gene was synthesized to accommodate the preferred codon usage for maize. The amy797E gene is regulated by the GZein promoter from Zea mays and the 35S terminator from the cauliflower mosaic virus.

The pmi gene represents the manA gene from E. coli and encodes the enzyme phosphomannose isomerase (PMI). It was used as a selectable marker gene during the transformation process. Mannose, a hexose sugar, is taken up by plants and converted to mannose-6-phosphate by hexokinase. This product cannot be further utilised in plants as they lack the PMI enzyme. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase, causing a block in glycolysis. It also depletes cells of orthophosphate required for the production of ATP. Therefore, while mannose has no direct toxicity on plant cells, it causes growth inhibition. This does not occur in plants transformed with the pmi gene as they can utilise mannose as a source of carbon. The pmi gene is regulated by the polyubiquitin promoter (ZmUbilnt) from maize and the NOS terminator from Agrobacterium tumefaciens.

Transformed cells were grown on cell culture media containing mannose and tested by PCR for the presence of both the amy797E and pmii genes and the absence of the spec gene (an antibiotic resistant marker in the plasmid backbone). Regenerated plants meeting these criteria were transferred to the greenhouse for propagation

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis was used to determine the insert and copy number of the amy797E and pmi genes and to confirm the absence of DNA sequence from outside the T-DNA borders of the transformation vector. Genomic DNA used for Southern blot analysis was isolated from pooled leaf tissue from ten plants representing the backcross four (BC4) generation of Event 3272 and 10 plants representing negative segregants. All plants used for genomic DNA isolation were confirmed for the presence and absence of amy797E and pmi genes using PCR. The Southern analyses demonstrated that event 3272 contains a single copy of the amy797E and pmi genes inserted as a single piece of T-DNA. The Southern analyses also confirmed the absence of DNA from the plasmid backbone.

To further characterise the integrity of the inserted T-DNA, the nucleotide sequence of the entire T-DNA insert was determined and compared to the DNA sequence of the transforming plasmid (pNOV7013). The final consensus sequence was determined by combining the sequence data from six individual clones to generate one consensus sequence for Event 3272. In total, 6100 bp of T-DNA was inserted into the corn genome. The consensus sequence data for Event 3272 T-DNA demonstrated the overall integrity of the insert and confirmed that the functional elements within the insert had been maintained. Sequence analysis revealed that some truncation occurred at the right and left borders of the T-DNA insert, most likely during the transformation process. The right border portion of the T-DNA insert was truncated by 23 bp and the left border portion by 7 bp. These deletions do not affect the normal function of the amy797E and pmi gene expression cassettes.

Genetic Stability of the Trait

Southern analyses were done to demonstrate that the insert in Event 3272 is stable over a number of generations. Genomic DNA for Southern analysis was isolated from pooled leaf tissue of ten plants each from three generations – BC1, BC2 and BC3 – and probed with an amy797E-specific probe. Negative segregants from the BC3 generation were used as controls. The hybridization pattern obtained with the amy797E-specific probe was found to be identical over the three generations examined.

The inheritance pattern of the T-DNA insert in Event 3272 was confirmed on individual plants from the BC1, BC2, BC3 and BC4 generations, analysed for the presence of the amy797E gene by TaqMan® PCR. The expected Mendelian inheritance ratio of positive and negative plants for a hemizygous trait in these populations is 1:1. The goodness of-fit of the observed genotypic ratio to the expected genotypic ratio was tested using Chi Square analysis, with Yates correction factor, and the hypothesis that the genetic trait is behaving in a Mendelian fashion was accepted for all generations.

Expressed material

The alpha-amylase activity of the AMY797E protein was compared to that of generic commercial Bacillus alpha-amylase using studies such as dose-equivalents, HPLC analysis of the size distribution of starch hydrolysis products, HPLC analysis of residual sugars and organic compounds, and ethanol production yield. These studies demonstrated that the AMY797E alpha-amylase is functionally equivalent in starch hydrolysis to other commercial alpha-amylases.

AMY797E protein was purified from grain of Event 3272 using a high temperature extraction process. Following separation of the extracted protein by SDS-PAGE, coomassie blue staining revealed a major band of an approximate molecular mass of 50.2 kDa (consistent with the predicted molecular mass of the mature AMY797E) and a minor low molecular mass band of The PMI protein produced in Event 3272-derived plants is encoded by the native pmi gene from E. coli (strain K-12). The protein consists of 391 amino acids and has a molecular weight of ca. 45 kDa. Plant cells expressing the pmi gene are capable of survival and growth in the presence of mannose as the only or primary carbon source. Under the same conditions, plant cells lacking PMI accumulate mannose-6-phosphate fail to grow. The PMI protein was extracted from leaf tissue from Event 3272 and its size, immunoreactivity, and specific enzymatic activity were compared to E. coli PMI. The PMI protein expressed in E. coli is identical in amino acid sequence to that encoded by the vector used to transform corn producing line 3272, with the exception of 16 amino acids added to the N-terminus as a result of the E. coli expression vector.

Samples of leaf extracts from Event 3272 and E. coli produced PMI were subjected to SDS-PAGE, followed by immunoblotting. A single immunoreactive band corresponding to a molecular mass of 42.8 kDa was detected in four separate leaf extracts. The E. coli-expressed PMI protein showed one major band with a slightly lower mobility compared to the plant expressed PMI, consistent with a slightly higher predicted molecular mass of 44.4 kDa (resulting from the additional 16 amino acids at the N-terminus). The mean enzymatic activity of PMI in leaf extracts ranged from 81.6 – 112.6 U/mg PMI, corresponding to an overall mean enzymatic activity of 96.9 ± 14.2 U/mg PMI. E. coli produced PMI had a slightly lower mean enzymatic activity of 52.9 ± 0.0 U/mg PMI. The two enzyme activities are comparable.

Mean AMY797E concentrations measured in grain from the maturity stage across four backcross generations were ca. 1044 - 1264 ?g/g fresh weight (fw; 1147 - 1389 ?g/g dry weight; dw). AMY797E levels were essentially undetectable in leaves and pollen although it was detected at low levels in the roots of some whorl stage plants (< 0.1 ?g/g fw).

Mean PMI concentrations in leaves from the anthesis stage across four backcross generations were ca. 6.6 - 9.3 ?g/g fw (25.6 - 35.7 ?g/g dw). PMI was detected at low levels in most of the plant tissues analyzed, except for some senescence-stage samples. Mean PMI levels measured in kernels at all developmental stages ranged from ca.

Environmental Safety Considerations Expand

Field Testing

Event 3272 maize hybrids were tested at 8 locations in 2003 and 17 locations in 2004 in the United States corn belt. A total of 26 agronomic traits were evaluated. These agronomic traits covered a broad range of characteristics that encompass the entire life cycle of the maize plant and included data assessing seedling emergence, vegetative vigour, basic morphology, growth habit, time to reproduction, susceptibility to European corn borer and gray leaf spot, and yield characteristics. Event 3272 is not expected to exhibit modified response to diseases and pest insects, compared to unmodified corn counterparts, as the expression of the AMY797E alpha-amylase and PMI phosphomannose isomerase is unrelated to plant pest potential. For the majority of agronomic traits, no statistically significant differences between Event 3272 hybrids and their non-transformed isogenic counterparts were observed.

Although instances of statistically significant differences between Event 3272 and control hybrids were observed for some traits, there were no consistent trends in the data across locations, hybrids or years, that would indicate that any of these differences were due to the genetic modification. Susceptibility to the European corn borer (ECB) could not be thoroughly evaluated due to very low ECB infestation levels. Similarly, susceptibility to Northern corn leaf blight, Southern corn leaf blight, and other diseases could not be evaluated due to a lack of naturally present disease inoculum. However, no change in response to pests or pathogens is anticipated from the expression of the AMY797E and PMI proteins in Event 3272. Furthermore, trial observations showed that susceptibility of Event 3272 hybrids to gray leaf spot disease is unchanged compared to control hybrids. The results showed no biologically meaningful differences between Event 3272 hybrids and their isogenic non-transgenic counterparts.

Outcrossing

The data included in the submission indicated that as pollen production and viability were unchanged by the genetic modification resulting in Event 3272, pollen dispersal by wind and outcropping frequency should be no different to other maize varieties. Gene exchange between Event 3272 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 and Canada, where there are no plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote.
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 or Canada, 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

Unmodified plants of Zea mays are not invasive of unmanaged habitats in the United States or Canada. Maize does not possess the potential to become weedy due to the lack of seed dormancy, the non-shattering nature of corn cobs and the poor competitive ability of seedlings. According to the information gained from the field trials, Event 3272 was determined to be similar to unmodified maize in this respect. No competitive advantage was conferred to Event 3272 by the expression of the AMY797E and PMI proteins. The introduction of these novel traits did not make Event 3272 maize weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified, and tolerance to disease was unchanged. It was concluded that Event 3272 has no increased weediness or invasiveness potential compared to currently commercialized corn varieties.

Secondary and Non-Target Adverse Effects

The AMY797E protein is not homologous to known toxins. A single-dose oral toxicity study showed that the AMY797E protein is not toxic to the mouse at a dose of 1,511 mg/kg body weight. This dose represents about 2.1 times the worst-case daily dietary dose for rodents eating a diet comprising 100% kernels of maize Event 3272 in the field. Alpha-amylases of varying degrees of amino-acid homology with AMY797E protein occur widely in nature among prokaryotes and eukaryotes. Alpha-amylase enzymes are present in plants, including maize. Alpha-amylase enzymes are also found in human saliva. There is an enormous diversity of alpha-amylases in soil microorganisms, including many heat-stable alpha-amylases. Therefore, it is likely that mammals, birds, insects and microorganisms exposed to AMY797E protein expressed in Event 3272 have had prior exposure to alpha-amylases. No harmful effects of such exposure is known.

Furthermore, as the AMY797E protein is not detectable in pollen of Event 3272, this maize poses no risk to pollinators and non-target pollen consumers. The kernel-specific expression of the protein in Event 3272, the lack of detectable mammalian toxicity of AMY797E, and the weight of evidence that alpha-amylases are not toxic to other wildlife, indicates minimal risk to non-target organisms resulting from exposure to AMY797E protein in Event 3272.

Phosphomannose isomerase is a ubiquitous enzyme involved in carbohydrate metabolism. Phosphomannose isomerases of varying degrees of amino acid homology to PMI expressed in Event 3272 occur widely in nature and have been detected in some crop species, mammals, humans, yeast, fungi and bacteria. Species that will be exposed to PMI from Event 3272 tissues are highly likely to have prior exposure to similar PMI proteins. No harmful effects of such exposure are known or expected. The PMI protein expressed in Event 3272 is not homologous to any known toxins and the bacterially-expressed PMI protein showed a lack of acute toxicity to the mouse at a single oral dose of 3,080 mg PMI/kg body weight. Based on this evidence, no adverse effects on organisms exposed to the PMI protein expressed in Event 3272 are anticipated.

Impact on Biodiversity

Event 3272 has no novel phenotypic characteristics that would extend its range beyond the current geographic range of maize production. Event 3272 is not more weedy or invasive than conventional maize and, since maize has no wild relatives with which it can outcross in the United States and Canada, there will be no transfer of the novel traits to other species in unmanaged environments. In addition, the novel traits were determined to pose minimal risk to non-target organisms and no changes to agronomic practices typically applied in management of conventional maize are required for Event 3272. Specifically, no increases in pesticides and fertilizers are required as well as no changes in cultivation, planting, harvesting or volunteer control. Thus, potential impacts on biodiversity are not expected to be different to the cultivation of conventional maize hybrids.

Food and/or Feed Safety Considerations Expand

Toxicity and Allergenicity

Corn line 3272 expresses two novel proteins – the enzymes AMY797E and PMI. AMY797E is expressed at relatively high levels in grain (838 –1627 µg/g fresh weight or 1004–3365 µg/g dry weight) and PMI is expressed at low levels (<0.4– 0.8 µg/g fresh weight or <0.5– 1.8 µg/g dry weight). Neither protein showed any evidence of toxicity in acute oral toxicity studies and also do not exhibit any amino acid sequence similarity with known protein toxins. Neither of the proteins is derived from sources known to be allergenic and both are rapidly digested in simulated digestion studies. AMY797E has no immunologically meaningful amino acid sequence similarity to known allergens. PMI was found to share a short sequence of amino acids (eight residues) with a known allergen, however further testing with human sera from allergic individuals did not demonstrate any cross-reactivity. Overall, the evidence indicates that AMY797E and PMI are non-toxic and have limited potential to be allergenic in humans.

Dietary Exposure

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. Given that the proposed use of this maize event is for the production of ethanol, is unlikely that the co-mingling of corn line 3272 with existing corn stocks will have any significant effect on population nutrition.

As the alpha-amylase enzyme expressed in Event 3272 has the ability to reduce starch molecules into component dextrins and mono/disaccharides at high temperatures, its potential impact on glycemic index, should Event 3272 be used as human food, was evaluated. This evaluation concluded that the co-mingling of Event 3272 with existing corn stocks is unlikely to have any significant effect on population nutrition. It is possible the AMY797E alpha-amylase may be activated during the cooking or processing of products containing Event 3272, which could increase the glycemic index of the final food product. However, even if the final food’s glycemic index was increased, the overall effect on the diet would be minimal, given that glycemic index is heavily influenced by other dietary factors.

Nutritional and Compositional Data

Compositional analyses were carried out to establish the nutritional adequacy of grain and forage from Event 3272, in comparison to conventional control lines. The constituents measured in grain from this event were protein, fat, carbohydrate, ash, moisture, fibre, fatty acids, amino acids, vitamins, minerals, secondary metabolites and anti-nutrients. In forage, the constituents measured were protein, carbohydrate, ash, moisture, fibre and minerals.

No compositional differences of biological significance were observed between the Event 3272 grain and forage and its non-GM counterpart. Several minor differences in key nutrients and other constituents were noted however the levels observed represented very minor differences and were within the reference range from conventional varieties. It can be concluded that food and feed from amylase-modified corn line 3272 is equivalent in composition to that from other commercial corn varieties.

A 42-day feeding study in broiler chickens supports this finding, with no evidence of biologically-significant differences in growth or feed conversion in chickens fed Event 3272 grain compared to near-isogenic control or commercially available maize lines.

Abstract Collapse

Maize line 3272 (OECD identifier SYN-E3272-5) contains two novel genes. The first, the amy797E gene, encodes the thermostable AMY797E alpha-amylase enzyme. alpha-Amylase catalyses the hydrolysis of starch by cleaving the internal alpha-1,4-glucosidic bonds into dextrins, maltose and glucose. The chimeric amy797E gene is derived from three wild-type alpha-amylase genes from the archael order Thermococcales. The amy797E gene encoded protein was selected for further development due to its thermostability, catalytic activity and reduced dependency on calcium under acidic conditions. These properties are important for the commercial application of alpha-amylase in the enzymatic hydrolysis of starch in dry-grind ethanol production from maize. The second gene, pmi (manA), derived from Escherichia coli, encodes the enzyme phosphomannose isomerase (PMI). The pmi gene is used as a selectable marker gene in the corn transformation process. Molecular and genetic analyses indicate that the transferred genes are stably integrated into the plant genome at one insertion site and are stably inherited from one generation to the next.

The majority of maize is grown for animal feed production and human food, however an increasing percentage is being used for the production of ethanol as a biofuel alternative to petroleum products from fossil sources. The hydrolysis of the large starch molecules in maize kernels is the first step in ehtanol production from maize and the themostable alpha-amylase in Event 3272 is intended to increase the efficiency of this process by operating at high temperatures. Although Event 3272 is not intended to be used directly for animal feed or human consumption, the residue remaining after starch hydrolysis and ethanol fermentation is used for animal feed, referred to as distillers dried grains with solubles (DDGS). There is also a possibility that grains of Event 3272 will enter the human food and animal feed chain through adventitious presence in other lots of maize grown for these purposes. As such, the event has been reviewed for both human food and animal feed safety, with no indications that there is any increased risk form inclusion of this event in the food and feed chains. One possible outcome, that the thermostable alpha-amylase may remain active during processing and hydrolyse starch thereby increasing the glycemic index of the final food product. However, even if the final food’s glycemic index was increased, the overall effect on the diet would be minimal given that glycemic index is heavily influenced by other dietary factors.

Event 3272 has been assessed for environmental impact and it was determined that there are no biologically meaningful differences between the event and isogenic non-transgenic counterparts with regards to plant pest potential, weediness or invasive potential. The novel proteins expressed in Event 3272 are not toxic in acute oral toxicity tests and show no homology to known toxins, indicating a minimal risk to non-target organisms in the environment exposed to the proteins in Event 3272. There are no phenotypic chracteristics in Event 3272 that would extend its range beyond that of conventional corn production and, together with the lack of any impact on non-target organisms, provides evidence to conclude that the potential impact of Event 3272 on the environment and biodiversity is equivalent to that of conventional maize varieties.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office

• Decision Document DD2008- 70: Determination of the Safety of Syngenta Seeds Inc.'s Corn (Zea mays L.) Event 3272
  [PDF Size: 165.94K bytes]

Food Standards Australia New Zealand

• Final Assessment Report: Application A580 - Food derived from amylase-modified corn line 3272
  [PDF Size: 545.66K bytes]

Health Canada Novel Foods

• Novel Food Information: Alpha-amylase corn event 3272
  [PDF Size: 64.34K bytes]

Japan (MAFF)

• Biological Diversity Assessment Report Thermostable α-amylase producing maize (Modified amy797E, Zea mays subsp. mays (L.) Iltis) (3272, OECD UI:SYN-E3272-5)
  [PDF Size: 195.97K bytes]

Philippines Department of Agriculture, Bureau of Plant Industry

• Determination of the Safety of Syngenta’s Corn 3272 (Corn for optimised bioethanol production) for Direct use as Food, Feed, and for Processing.
  [PDF Size: 40.56K bytes]

U.S. Department of Agriculture, Animal and Plant Health Inspection Service

• Availability of Petition and Environmental Assessment for Determination of Nonregulated Status for Corn Genetically Engineered To Produce an Enzyme That Facilitates Ethanol Production [Docket No. APHIS–2007–0016]
  [PDF Size: 55.84K bytes]
• Petition for nonregulated status of corn event 3272
  [PDF Size: 1.24M bytes]

United States Food and Drug Administration

• Biotechnology Consultation Note to the File BNF No. 000095
  [PDF Size: 52.86K bytes]

This record was last modified on Wednesday, November 9, 2016