Question: Can the effects of such directed gene shuffling be accomplished without direction? If so, what’s the evidence?
Gene Shuffling to Produce Food and Feed Crops
Prof. Joe Cummins
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 Ã¢â‚¬Å“improvedÃ¢â‚¬Â 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 Ã¢â‚¬ËœcloudÃ¢â‚¬â„¢ 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 Ã¢â‚¬Å“the applications of gene shuffling are multipleÃ¢â‚¬Â (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 Ã¢â‚¬Å“rationalÃ¢â‚¬Â enzyme redesign (14). Chemistry has a long history of creating Ã¢â‚¬Å“coolÃ¢â‚¬Â processes for creating products heedlessly without consideration of potential hazards to humans and the environment. Ã¢â‚¬Å“RationalÃ¢â‚¬Â 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 Ã¢â‚¬Å“rationalÃ¢â‚¬Â design.
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 Ã¢â‚¬Å“gene shufflingÃ¢â‚¬Â technologyÃ¢â‚¬Â Press Release 2005
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