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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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