In the early 1990′s, a number of molecular biologists became interested in generating novel proteins in vitro by duplicating the evolutionary process in their laboratories. In a bid to do so, they resorted to in vitro mutagenesis in order to improve the properties of enzymes in a stepwise fashion. Since mutations caused by artificial treatments are random and frequently deleterious, a large population was necessary for screening before an improved strain or variety of a plant or an animal could be obtained.
Since this initial approach, technology has improved and the old process has been replaced by a new technique called DNA shuffling. In a review article published in the April, 2001 (4:152-156) issue of “Current Opinion in Plant Biology”, Dr. Michael Lassner and John Bedbrook at Maxygen Incorporated, California, elaborate on the new technology, its modus operandi, its scope for further improvement and its uses in the generation of novel carotenoids, enhanced detoxification and the improvement of insect resistance genes.
In the DNA shuffling technique, variants of an individual gene or a set of homologous genes are first fragmented and then recombined to give rise to genes of different base compositions. In the reassembly process, fragments act as primers for each other. The recombinant genes are then screened for the desired improvements in the properties of a target enzyme. In order to further increase the efficiency of a synthetic enzyme, the process is repeated; in the second and subsequent rounds of DNA shuffling, the best progeny from the previous round are used as parents to continue the process of improvement. DNA shuffling has an added advantage in that using different DNA polymerases can control the rate of random mutagenesis. The authors illustrate this process with an example showing how the enhancement of unexpected components brought about improvement of an arsenate degradation pathway.
The authors provide the following antibiodic example to illustrate the advantages of multi-gene shuffling over that of single gene variants. After intragenic shuffling, an E. coli strain grew on a medium with 8-fold more moxalactam, (an antibiotic). In contrast, using a DNA shuffling of four parents, the technique yielded a recombinant that showed a 270-fold improvement over the most active parent in their ability to grow in the above antibiotic.
The authors emphasize that for any gene improvement program, there must be a suitable assay system to identify gene variants. They describe a clonal selection in which libraries of gene variants are created by shuffling 26 parents. Variants expressing inactive proteins were discarded. Thereafter, a library of 654 active clones was tested for 5 properties. Improved variants showing improvement in 1 or more characters were detected. A few showed improvements in all 5 characters used as test criteria.
Carotenoid biosynthesis: Plants utilize two desaturase enzymes to produce the red carotenoid, lycopene. The latter is acted upon by lycopene cyclases to form various forms of carotenes. In the next chain of chemical reactions, ketolases and two other enzymes act upon these substances to synthesize oxygenated carotenoids called xanthophylls. DNA shuffling technology has been used to produce a novel carotenoid compound with improved antioxidant properties. The authors give an example of one gene variant capable of synthesizing between 2-6 double bonds i.e. 3, 4, 3′, 4′ tetradehydrolycopene, a hitherto unknown natural compound with greater antioxidant properties. Quoting some more examples, the authors predict that using DNA shuffling technology, it may be possible to create more powerful enzymes capable of producing enhanced levels of a specific compound. In this connection the authors suggest that the present shortfall of the ‘Golden Rice” in its lack of ability to synthesize sufficient amount of beta-carotene can be overcome by tailoring the specific enzymes using DNA shuffling technology to raise the level of beta carotene.
It has also been shown that the use of this technology has led to the development of recombinant enzymes capable of hydrolyzing triazine to non-toxic compounds not only at a greater efficiency level but also degrading related triazine compounds not amenable to biodegradation by any known bacterial population. The authors suggest that construction of such novel enzymes could be used to remove toxic triazines and related pollutants from soil and groundwater.
The authors provide examples of using DNA shuffling to enhance the potency of a natural biotoxins. Screening approximately 4,000 gene variants led to the detection of a BT line with EC50 (concentration of toxic protein which kills half of the insects) 3.8 more efficient over the parent. In another gene-shuffling experiment, some highly insect resistant gene variants were found: of these some were 6.7 times more active than the cry1CA parent against Spodoptera exigua and others three times more efficient than cry1AB against Heliothis zea. What was more interesting is that among the BT variants, there were some that were found active against both the insects. The results indicate that the spectrum of BT-controlled insect resistance can be widened by DNA shuffling. For insect control, this is definitely a significant technological advancement that can be used for the production of next generation transgenic plants.
DNA shuffling also appears to be a highly effective tool for the production of enzymes that are tailored for specific uses. Directed molecular evolution can be used to improve enzymes or pathways according to the molecular breeder’s objectives.
The authors point out that despite the similarities of directed molecular evolution with conventional plant and animal breeding, there are also major differences. In conventional breeding, only two parents are involved in hybridization, whereas in DNA shuffling there is a simultaneous recombination of multitude of parents. As against selection for 1-3 pairs of characters, in DNA shuffling technology one can focus selection for the improvement of proteins in a variety of ways. The most convenient thing about this recent technique is that DNA shuffling and screening can be accomplished in a matter of weeks as against a few to many years depending on the life cycle of the plant in question. Most certainly, this strategy will be in use more and more in the coming years for improving the agricultural products of both animal and plant origin.