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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:
http://www.regulations.gov/fdmspublic-bld61/component/main
References
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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