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[SANET-MG] high lysine maize

October 14, 2005

Prof. Joe Cummins

High Lysine Maize

Maize is a domesticated grass of tropical Mexican origin. Maize is the third most planted crop after wheat and rice. The largest producers of maize are the United States, China and Brazil. Maize evolved with humans and depends on humans for its very existence because the seeds require humans for dissemination from the cobs. Maize is mainly produced for food , feed and fodder. The main kinds of maize include flint which makes up about 14% of world production, flint has a very hard seed coat suitable where storage and germination conditions are poor. Flour maize is the preferred form for human consumption in tortilla, dumpling, or for direct consumption. It accounts for 12% of commercial production. Dent has a characteristic dent in the dry kernel, it is used as livestock feed, starch, syrup, oil and alcohol. 73% of world production of maize is dent. Sweet corn is canned , frozen or served fresh to humans , it along with pop corn make up about 1% of world production of maize (1). Genetically modified (GM) maize has been incorporated into the different kinds of maize by crossing them with the original GM maize.

Maize is a major source of food and feed worldwide but it is not a suitable single source of nutrition. Maize alone does not provide the essential amino acid , lysine, in sufficient quantity for the nutritional needs of humans and for farm animals. Traditional maize diets were accompanied by dry beans to compensate for the deficiency of lysine in maize. Currently, maize flour and feeds are supplemented with lysine in areas of the world where the supplement can be afforded. The Food and Agriculture Organization of the United Nations has reviewed the nutritional deficiency in conventional maize and established requirements for the amino acid (2,3). Daily requirements for lysine have been set at 400 to 900 mg for men and 300 to 700 mg for women but benefits were gained when lysine was increased to 1800 mg per day then more gradually up to 3600 mg (4). Interestingly, piglets discriminated between diets rich and poor in lysine and selected the lysine rich diets (5).

Efforts to increase maize lysine using traditional breeding have been pursued for many years. In 1964 the opaque2 mutants were found to produce elevated levels of. Lysine. That mutant increases production by decreasing content of zein storage proteins allowing lysine rich proteins to accumulate in the endosperm, The original mutants were unsuitable for commercial production but introducing a battery of modifier genes that improved the field and storage qualities of the maize. Improvements using quantitative trait loci are allowing improvements in maize allowing production of high lysine maize in countries where it can impact human nutrition (6).

The mutant high lysine strains selected for conventional breeding have dealt with the production of proteins richer in lysine than the existing lines, Monsanto Corporation developed a maize line that employs a synthetic approximation of a bacterial gene to increase the level of lysine in cellular amino acid pools in the grain. That transgenic maize line is the basis of a petition for non-regulated status in the United States. The maize line constructed by Monsanto maize LY038 was developed, through the use of recombinant DNA techniques, to integrate the cordapA coding sequence into the maize genome. The cordapA sequence is under the control of the maize Glb1(Globulin 1) promoter to direct expression of the Corynebacterium glutamicum-derived lysine-insensitive dihydrodipicolinate synthase (cDHDPS) enzyme predominantly in the embryo, to increase the level of lysine in grain for animal feed applications. The cordapA sequence driven by the Glb1 promoter was preceded by two synthetic linker sequences , an intron from rice actin gene and a chloroplast targeting sequence from maize DHPDS gene. In the primary construct there was included a lox P (recombination site recognized by Cre recombinase) followed by a CaMV promoter driving a neomycin antibiotic resistance gene along with bleomycin resistance gene with a nos transcription terminator from Agrobacterium. Finally, an ampicillin resistance gene with bacterial promoter was included according to Table IV-1 page 34 of the petition (7). The basic idea of the construction was to introduce a bacterial enzyme that had a relaxed feed back inhibition on lysine level allowing lysine to accumulate in the cellular amino acid pool.

The purpose of the lox recombination sites was to provide a means of removing the neomycin antibiotic resistance gene after it use in selection was no longer required (It is not clear why the ampicillin resistance gene was allowed to remain in the final strain). The Cre recombinase acts at the lox sites to remove the neomycin resistance gene cassette. Plants expressing the gene for Cre recombinase are prone to phenotypic aberrations (8), while DNA damage and growth inhibition is observed in mammalian cells treated with Cre recombinase (9). To avoid those complications a maize stain bearing the Cre recombinase gene was crossed with the high lysine transgenic maize line to remove the neomycin cassette. When it was established that the line lacked the neomycin cassette the high lysine maize-Cre recombinase hybrid line was selfed and by the F3 generation plants lacking the Cre-recominase gene were selected and used to establish the final high lysine maize line(7).

The leading question is transgenic high lysine more economically advantageous over amino acid supplementation or use of high lysine strains produced by conventional breeding? An analysis reported in a FAO workshop indicated that lysine supplementation was far more economical source of lysine than was transgenic high lysine maize (10). The conventional bred high lysine maize has also gone much further to accommodate the needs of indigenous farmers for high lysine maize (6).

There did not appear to have been a full sequence of the transgenic DHPDS protein nor was there a search for allergenic epitopes in the protein structure. There did not appear to be any feeding experiments with the transgenic DHPDS protein ,or for that matter, with the transgenic high lysine maize (7). Finally, effort should be made to determine whether or not the lysine stored in cellular pools portrays stability and availability equivalent to the stored lysine rich proteins during processing for food or feed. The petition for non-regulated status should not be granted until these matters are cleared up.

Public Comment on the Monsanto Proposal for High Lysine Maize can be made until

28 November 2005 at URL:



1.Salvador,R. Maize 2005 http://maize.agron.iastate.edu/maizearticle.html

2.FAO Maize in human nutrition FAO Document Repository 1992 http://www.fao.org/documents/

3. FAO Protein Sources for the Animal Feed Industry 2002 http://www.fao.org/documents/

4. Clark HE, Bailey LB. and Brewer MF. Lysine and tryptophan in cereal-based diets for adult human subjects. Am J Clin Nutr. 1977 May;30(5):674-80

5.Kirchgessner,M,Stangl,G. and Roth,F. Evidence for specific dietary selection for lysine by the piglet J. Anim. Physiology and Anim. Nutr. 1999 81,124-31

6. Gibbon,B. and Larkins,B. Molecular genetic approaches to developing quality protein maize Trends in Genetics 2005 21, 227-33

7.Lucas,D. Petition for determination of nonregulated status for lysine maize LY038 USDA/APHIS 2004 http://www.aphis.usda.gov/brs/aphisdocs/04_22901p.pdf

8. Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJ, Nijkamp HJ and van Haaren MJ. Cre recombinase expression can result in phenotypic aberrations in plants Plant Mol Biol. 2003 Jan;51(2):263-79

9. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R, Berns A and Jonkers J. Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9209-14

10. Toride,Y. Lysine and other amino acids for feed production and contribution to protein utilization in animal feeding FAO Protein Sources for the Animal Feed Industry 2002 http://www.fao.org/ag/aga/workshop/feed/papers/12yashiko.doc.

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