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[SANET-MG] gene shuffling to created food and feed
January 12, 2006
Prof. Joe Cummins
Gene Shuffling to Produce Food and Feed Crops
Gene shuffling is a process consisting of producing a large array of
producing many variants of a DNA sequence from a gene or a portion of a
gene representing the an active site on a protein then reassemble the
gene from random fragments using recombination. The recombinants are
cloned into a vector then 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 proteins obtained from living organisms.
MaeWan Ho has criticized gene shuffling because it is inherently
dangerous it is likely to produce viruses, bacteria or eukaryote forms
with unexpected and malevolent toxicity because recombination is the
basis for the appearances of novel devastating pathogens (1).In spite of
these concerns gene shuffling has been burgeoning and has been described
as directed evolution or rational design, promoters of the technique
sometimes ascribe the technique to be a simple extension of the work of
Charles Darwin while others equate it to intelligent design Recently the
giant seed company Pioneer Hybrid (a subsidiary of DuPont Chemical )
announced that they had applied gene shuffling to produce a gene that
coded a protein 2000 times more potent than a natural glyphosate
(herbicide) resistance gene(2). The research coordinator in charge of
gene shuffling at Pioneer is Lynda Castle who was among those applying
for a patent on DNA shuffling to produce herbicide selective crops
associated with Maxygen,Inc. Redwood City, California (3).
The synthetic gene designed to inactivate the herbicide glyphosate may
be the first crop gene released for commercial application that was
altered using gene shuffling. The gene enhanced using DNA shuffling was
isolated from the bacterium Bacillus licheniformis , the gene codes an
enzyme that inactivates the herbicide by adding an acetyl group at the
nitrogen atom in glyphosate. Using gene shuffling the activity of the
enzyme by four orders of magnitude (4,5). Glyphosate inhibits
5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS), a nuclear-encoded
chloroplast-localized enzyme ( the shikimic acid pathway of plants and
microorganisms.is the pathway producing aromatic amino acids) and most
commercial glyphosate tolerant crops are based on insensitivity of
EPSPS. Inactivation of glyphosate by acetylation should prevent the
herbicide from accumulating in food and feed crops as it does in the
other 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).
The diphenylether herbicides (flurodifen for example) are used
extensively with food crops. The diphenylether herbicides are detoxified
by an enzyme glutathione transferase which adds glutathione to the
herbicide. Using a maize tau class glutathione transferase enzyme from
maize and a process called forced evolution (gene shuffling and
reconstructive error prone PCR) genes specifying glutathione transferase
enhanced flurodifen inactivating activity were created (6).Forced
evolution herbicide tolerant crops have not yet been submitted for
commercial release but they should soon be ready for commercialization.
Many questions remain unanswered about the human and environmental
safety of the synthetic genes.
Plant virus vectors have been “improved” using mutagenesis and gene
shuffling. The expression vectors are used to produce therapeutic
proteins and to assist in screening plant genes to determine their
function. A tobacco mosaic virus vector was modified to extend host
range and to enhance movement within plants using DNA shuffling and
mutagenesis (7). The serious consequences flowing from recombination
between the modified vector and wild plant viruses was hardly discussed
yet those consequences could be profound and they are predictable. Plant
virus vectors such as tobacco mosaic virus and barley stripe mosaic
virus are beginning to be used extensively as epigenetic gene expression
systems useful in gene discovery or for producing quantities of valuable
pharmaceutical protein. The plant virus vector systems is an attractive
tool for screening and producing genes and proteins derived from
shuffled genes. However, the consequences of introducing shuffled genes
into a pool of native virus is bound to be dire. An RNA virus population
does not consist of a single genotype; rather, it is an ensemble of
related sequences, termed quasispecies. Quasispecies arise from rapid
genomic evolution powered by the high mutation rate of RNA viral
replication.
Although a high mutation rate is dangerous for a virus because it
results in nonviable individuals, it has been hypothesized that high
mutation rates create a ‘cloud’ of potentially beneficial mutations at
the population level, which afford the viral quasispecies a greater
probability to evolve and adapt to new environments and challenges
during infection(8).Viral vectors may easily form quasispecies within
virus populations and exert highly detrimental effects upon infection of
hosts.
DNA shuffling has been effective in exploring gene function as in the
analysis of a tomato gene, Pto, conferring multiple disease resistance
(9). Studies of that type are a first step towards creation and testing
of commercial modified plants. DNA shuffling was used to study nitrogen
fixation in Sinorhizobium-alfalfa symbiosis (10). Genetically modified
Synorhizobium are already extensively released in North America ,
bacteria enhanced using gene shuffling create a major concern. Protease
inhibitor genes to control plant nematode parasites are being enhanced
using gene shuffling but successful results have not yet been released (11).
The Encyclopedia of Chemical Processing included a chapter on Plant
Metabolic Engineering. That chapter discussed gene shuffling and
directed enzyme evolution indicated that “the applications of gene
shuffling are multiple” (12). The chapter did not hint that gene
shuffling may lead to untoward consequences and should be approached
with caution. Chemists seem to have taken biocatalysts to heart. A
review , Directed Evolution and Biocatalysts, stresses the utility of
designer enzymes in industrial chemistry (13). As in most of the
chemical industry publications the possible pitfalls and hazards of gene
shuffling are ignored. A review, Enzyme Redesign, shows the extent to
which chemistry has taken gene redesign to heart with 165 journal
references to “rational” enzyme redesign (14). Chemistry has a long
history of creating “cool” processes for creating products heedlessly
without consideration of potential hazards to humans and the
environment. “Rational” design seems rather irrational, at best.
Those who express concern about synthetic genes shuffled to create
commercial products are beginning to look like the Dutch boy who stuck
his finger in a small hole in the dike to prevent a great flood.
However, we must continue to try to provide some rational discussion for
“rational” design.
References
1.Ho,MW Death by DNA shuffling Science in Society 2003,18, 9
2.Pioneer Hi-Bred International Inc. Pioneer breaks new ground for trait
optimization with “gene shuffling” technology” Press Release 2005
http://www.pioneer.com/pioneer_news/press_releases/products/trait_opt.htm
3. Subamanian,V, Stemmer,W. ,Castle,L.,Muchhal,U. and Siehl,D. DNA
shuffling to produce herbicide selective plants 2002 United States
Patent Application 20020058249
4. Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, Bertain S, Cho HJ,
Duck N, Wong J, Liu D. and Lassner MW. Discovery and directed evolution
of a glyphosate tolerance gene. Science. 2004 May 21;304(5674):1151-4
5. Siehl DL, Castle LA, Gorton R, Chen YH, Bertain S, Cho HJ, Keenan R,
Liu D and Lassner MW. Evolution of a microbial acetyltransferase for
modification of glyphosate: a novel tolerance strategy. Pest Manag Sci.
2005 Mar;61(3):235-40
6. Dixon DP, McEwen AG, Lapthorn AJ and Edwards R. Forced evolution of a
herbicide detoxifying glutathione transferase. J Biol Chem. 2003 Jun
27;278(26):23930-5
7. Toth RL, Pogue GP and Chapman S. Improvement of the movement and host
range properties of a plant virus vector through DNA shuffling. Plant J.
2002 Jun;30(5):593-600
8. Vignuzzi M, Stone JK, Arnold JJ, Cameron CE and Andino R.
Quasispecies diversity determines pathogenesis through cooperative
interactions in a viral population. Nature. 2006 Jan 19;439(7074):344-8
9. Bernal AJ, Pan Q, Pollack J, Rose L, Kozik A, Willits N, Luo Y,
Guittet M, Kochetkova E and Michelmore RW. Functional analysis of the
plant disease resistance gene Pto using DNA shuffling. J Biol Chem. 2005
Jun 17;280(24):23073-83
10. Grzemski W, Akowski JP and Kahn ML. Probing the Sinorhizobium
meliloti-alfalfa symbiosis using temperature-sensitive and
impaired-function citrate synthase mutants. Mol Plant Microbe Interact.
2005 Feb;18(2):134-41
11. McPherson MJ and Harrison DJ. Protease inhibitors and directed
evolution: enhancing plant resistance to nematodes. Biochem Soc Symp.
2001;(68):125-42
12. Wurtzel,E. and Grotewald,E. Plant metabolic engineering Encyclopedia
of Chemical Pocessing 2006 Taylor & Francis DOI: 10.1081/E-ECHP-120039300
13. Powell KA, Ramer SW, Del Cardayre SB, Stemmer WP, Tobin MB,
Longchamp PF and Huisman GW. Directed Evolution and Biocatalysis. Angew
Chem Int Ed Engl. 2001 Nov 5;40(21):3948-3959
14.Penning,T. Enzyme redesign 2001 Chem.Rev. 101,3027-46
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