Until now researchers in the field of bioengineering and plant breeding have altered the expression of existing enzymes or have introduced the expression of a phy-tochemical. These approaches have in common the fact that no novel phytochemicals are synthesized; no "new to nature" products have been made. New theories and technologies are being developed that are directed toward changing the efficiency of enzymes, and in these experiments, phytochemicals have been produced in microorganisms that are "essentially inaccessible from natural sources or by synthetic chemistry" (Schmidt-Dannert et al., 2000).
This recent body of work applies the principles of Darwinian evolution to alter enzyme efficiency and function (Arnold and Volkov, 1999; Stemmer, 1994a,b). Two separate strategies have been utilized; a rational design approach involving iterative computer protein design and site-directed mutagenesis of single nucleotides, and a second strategy in which blocks of nucleotides are shuffled to produce novel pep-tides, a method called "DNA shuffling" (Stemmer, 1994,a,b). The former method can be viewed as in vitro molecular evolution because in vitro recombination can occur between the DNA templates of two related genes that differ in their sequence, and the recombinants screened for improved performance (Stemmer, 1994,a,b). The DNA templates can contain random point mutations, single genes, or homologous genes (Crameri et al., 1998).
As mentioned above, a frequently used method in plant breeding is the back-cross, in which a specific trait is introduced into a cultivar or variety and the desired genetic background is recovered by repeated crosses of the progeny to the recurrent parent. Similarly, by molecular backcrossing against the wild-type DNA, nonessen-tial mutations can be selected against in the resulting recombinants (Stemmer, 1994). This relatively simple technique resulted in a 32,000-fold increase in antibiotic resistance (Stemmer, 1994a), and a 1,000-fold increase in substrate specificity of P-fucosidase (Zhang et al., 1997). Related sequences have been bred (Schmidt-Dannert et al., 2000), as well as sequences with only 50% homology (Ostermeier et al., 1999). One can imagine a strategy to improve metabolic processes in plants using in vitro molecular evolution.
To increase the efficiency with which candidate plants and phytochemicals are discovered, more sophisticated networks are being developed between ethnob-otanists, medical professionals, and the agriculture/botany community. As the process matures, increasingly sophisticated experiments can be designed to more clearly define the role of phytochemicals in human health and plant function. Cell lines from specific tissues and defined genotypes can be used to screen the efficacy of phytochemicals. Genetic and metabolic profiling of plants will enable phyto-chemicals to be precisely identified and determine under what conditions they are produced. Information from cell screening can be used to direct the development of plants with unique phytochemical profiles. Any promising plant collected from the wild must be adapted to agriculture, and its propagation methods worked out. Most perennial, and many annual plants have seed dormancy mechanisms that make efficient agronomic production all but impossible. Further, the seed of many subtropical and tropical plants are recalcitrant, meaning they cannot withstand desiccation and therefore cannot be stored for appreciable lengths of time. Alternatively, whole metabolic pathways, or portions thereof, could be transferred into domesticated plants exhibiting good agronomic characteristics such as high biomass production and no seed dormancy.
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