Engineered with novel procedures and constructs for multiple resistances to toxic herbicides Now seeking non-regulated status from obliging regulators. Prof. Joe Cummins and Dr. Mae-Wan Ho
This report has been submitted to the United States Department of Agriculture (USDA) on behalf of I-SIS
The United States Department of Agriculture Animal and Plant Health Inspection Service(USDA/APHIS) has received a petition from Pioneer Hi-Bred International, Inc., seeking non-regulated status for soybean designated as transformation event 356043, which has been genetically engineered for tolerance to glyphosate and acetolactate synthase-inhibiting herbicides. The docket providing background information and instructions for commenting on the proposal, designated APHIS-2007-0019, is located at: http://www.regulations.gov/fdmspublic/component/main. Public comments are due by 4 December 2007.
Pioneer’s 356043 soybean has been genetically engineered to express modified glyphosate acetyltransferase (GAT46014) and acetolactate synthase (ALS) proteins. These genes impart tolerance to both glyphosate and ALS-inhibiting herbicides (e.g., sulfonylureas and imidazolinones). The gat46014 gene is a synthetic recombinant of a gene derived from a common soil bacterium Bacillus licheniformis, but artificially improved for activity by DNA shuffling (see below). Its expression is driven by a synthetic promoter (SCP1). The gene conferring tolerance to ALS-inhibiting herbicides is gm-hra, a modified soybean als gene, and its expression is driven by a soybean promoter, also artificially enhanced. A single copy of these genes and other DNA regulatory sequences were introduced into soybean somatic embryos using microprojectile bombardment [1, 2].
Soybean 356043 involves a novel, untried technique called DNA- or gene-shuffling to create a synthetic gene that inactivates the herbicide glyphosate ten thousand times more effectively. In this process, many variants of a DNA sequence from a gene or a portion of a gene are chopped into small fragments and allowed to reassemble at random to generate numerous recombinants  (see Death by DNA Shuffling, SiS 18). The original variants are created using directed mutation of selected codons, or error prone gene replication. The numerous recombinants resulting from DNA shuffling are cloned into a vector and introduced into bacteria or virus for rapid screening. Using this technique it has been possible to produce genes for enzymes, regulatory proteins or antibodies with laboratory performance far superior to the natural proteins obtained from living organisms. Unfortunately, the technique is inherently dangerous, as it is likely to produce variants with unexpected and unknown toxicity or immunogenicity. As DNA shuffling can produce billions of recombinants in a matter of hours, it is unlikely that most of the recombinants will be characterized or identified. In spite of these concerns, gene shuffling has been burgeoning. Promoters of the technique sometimes describe the technique as a simple extension of the work of Charles Darwin, or equate it to “intelligent design”. Pioneer Hybrid (a subsidiary of DuPont Chemical) announced that they had applied gene shuffling to produce a gene that encodes a protein 2000 times more potent than a natural glyphosate (herbicide) resistance gene . The research coordinator in charge of gene shuffling at Pioneer, Lynda Castle, was among those applying for a patent on DNA shuffling to produce herbicide tolerant crops when she was associated with Maxygen Inc. Redwood City, California . The synthetic gene designed to inactivate the herbicide glyphosate may be the first crop gene released for commercial application that has been created using gene shuffling.
The gene gat46014 enhanced using DNA shuffling, isolated from the bacterium Bacillus licheniformis, codes for an enzyme that inactivates the herbicide by adding an acetyl group at the nitrogen atom in the glyphosate molecule. Gene shuffling has enhanced the activity of the native enzyme by four orders of magnitude [6-9]. What are the health and environmental impacts of this new synthetic protein? This question has not been addressed.
It should be noted that all transgenic proteins may be immunogenic, even when otherwise harmless proteins are transferred to a closely related species [10, 11] (Transgenic Pea that Made Mice Ill, SiS 29).
Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a nuclear-encoded enzyme that is transported into the chloroplast (the shikimic acid pathway of plants and microorganisms is responsible for producing aromatic amino acids), and most currently grown commercial glyphosate tolerant crops are based on an EPSPS enzyme that is insensitive to glyphosate. Inactivation of glyphosate by acetylation should prevent the herbicide from accumulating in food and feed crops as it does in the current glyphosate tolerant crops and be safer provided that acetylated glyphosate is found to be non-toxic to animals (safety evaluations of the compound are not presently available) and that there is no possibility of de-acetylation within the animals.
The Pioneer petition , the USDA/APHIS environment assessment  and a peer reviewed publication  report on a 42 day feeding experiment with soybean 356043 on broiler chickens. They reviewed measurements of broiler weight and organ size but did not include complete necropsy studies for possible changes in the cells and tissues of the treated animals. Acetylated glyphosate certainly accumulates in animals and humans eating modified soybeans but the Pioneer petition  and USDA/APHIS environment assessment  argued that as acetylated amino acids and proteins are not known to be toxic to animals and humans, acetylated glyphosate herbicide will not be toxic either, which seems a highly fallacious and unjustified extrapolation. There has been no direct study of the potential toxicity of acetyl glyphosate, nor of the transgenic protein. Furthermore, the possibility that de-acetylation of glyphosate may occur within the digestive system and cells of the organism has not been considered.
Soybean event 356043 has been constructed by transforming the plant using a plasmid containing two gene cassettes. The first cassette contains the synthetic glyphosate acetyl transferace (GAT) gene. The second cassette contains the modified acetolactate (ALS) gene to confer resistance to multiple herbicides (e.g., sulfonylureas and imidazolinones). The GAT gene is driven by a novel synthetic promoter derived from a portion of the CaMV promoter and a core consensus promoter (Rsyn7-SynII). Synthetic promoters have been developed for crop use by Pioneer HyBred company  and the herbicide tolerant 356043 soybean is the first genetically modified crop to be proposed for commercial release containing both synthetic promoters and synthetic structural genes. While synthetic structural genes already raise serious safety concerns, synthetic promoters are even more problematic, as they involve higher-level regulations that extend to multiple genes and multiple sets of genes. The GAT synthetic promoter is further followed by a tobacco mosaic virus translation enhancer sequence, a novel combination that has also been completely untested for safety. Similarly, the gm-hra gene cassette contains a promoter from the soybean S-adenosyl –L-methionine synthetase gene (SAMS gene) followed by an intron gene from the SAMS gene, another novel untested combination . These synthetic constructs, especially synthetic promoters are completely untested for safety.
Particularly worrying is the spread of these synthetic promoter and constructs by horizontal gene transfer and recombination, with the potential of creating new viral and bacteria pathogens, and in the case of animals and human cells, the strong synthetic promoters may trigger cancer if inserted next to protooncogenes. We have been warning of the dangers of horizontal gene transfer in numerous articles (see for example, Horizontal Gene Transfer - The Hidden Hazards of Genetic Engineering , ISIS Report; and most recently, GM Food Nightmare Unfolding in the Regulatory Sham )
USDA/APHIS has completed ignored recent evidence on the serious toxicity of glyphosate  (Glyphosate Toxic & Roundup Worse , SiS 26) and its lethality to frogs [17, 18]. Similarly, ALS-inhibiting herbicides are known to be acutely toxic for aquatic micro-organisms and other plants and animals living on or around surfaces of submerged rocks at nanomolar concentrations [19, 20].
There is no case for the release of soybean 356043 to the environment, let alone granting it unregulated status. To do so will be making an utter mockery of regulation.
This is number 39 of ISIS submission to USDA.
Article first published 30/11/07
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