Using the technologies of laboratory-directed evolution and applying the methods of chemical engineering to devise efficient and robust high-throughput screens for enzyme evolution offer the promise to revolutionize biological transformations.
Input traits could be significantly improved via enzyme engineering. For instance to improve insect resistance, it may be possible to recombine protective proteins such as Bacillus thuringiensis toxin (BT) from multiple independent sources to create novel variant BT proteins with either increased potency, or decreased ability to induce resistance in the targeted pest. Alternatively, it may be possible to improve the efficiency of various pathway enzymes to synthesize more of a particular protective compound, or changing the chirality of an individual protective compound.
Output traits present the most easily defined targets for plant improvement. Plants synthesize a bewildering array of secondary products that have uses ranging from chemical feedstocks to foodstuffs to pharmaceuticals. By enzyme engineering, it may be possible to improve the accumulation of desired metabolites. Plants can efficiently convert CO2, one of the only natural resources that continues to become more abundant, into reduced carbon storage compounds using sunlight as the energy source. It is easy to imagine replacing the enzymes and pathways used to synthesize storage proteins, carbohydrates, and lipids to novel pathways to make and store just about any organic molecule we can conceive. For example, three enzyme pathways for the accumulation of novel polyhydroxyalcanoates have been successfully engineered into plants (Poirier, 2001). Because plant oils are relatively inexpensive to produce, pathways designed to produce modified oils with desirable properties as industrial feedstocks are particularly attractive (Thelen and Ohlrogge, 2002).
Many of the natural enzymes with novel function in pathways such as fatty acid biosynthesis have been identified. However, alteration of biochemical regulation of enzyme activity via enzyme engineering of protein stability, sites of posttranslational modifications, and of allostery represents underexploited opportunities in plant biotechnology.
Allosteric regulation involves the positive or negative modulation of enzyme activity after binding of one or more metabolite(s). It represents a particularly interesting enzyme-engineering target in that the introduction of an enzyme with altered sensitivity to the interacting metabolite can overcome a potent metabolic block that cannot be overcome by simply controlling the abundance of the enzyme. In addition, for many cases, the introduction of an allosterically insensitive variant enzyme should overcome the metabolic block even in the presence of the endogenous allosterically sensitive enzyme. Several strategies can be used to identify enzymes with altered regulation. The first is to identify a naturally occurring enzyme from a source that does not exhibit allosteric regulation and to introduce the corresponding gene into the desired host organism. The second is to perform enzyme engineering and activity screening to identify variants in which the catalytic activity of the enzyme is maintained, but in which the binding of the allosteric regulator is disrupted. An excellent example of overcoming allostery involves starch metabolism. A nonregulated mutant of the E. coli ADPG pyrophosphorylase enzyme was identified and introduced into potato tubers (Ballicora et al., 2003), resulting in a 25–60% increase in accumulation of starch compared to tubers containing the wild-type enzyme (Preiss, 1996). It is possible that under certain conditions, the metabolic flux into the desired endproduct may not substantially increase if the allosterically regulated step was either colimiting or not limiting to the rate of product accumulation. In these cases, metabolic profiling (Graham et al., 2002) can be employed to identify the new rate-limiting step, and efforts to increase the activity of this step can be undertaken. Similar approaches can conceivably be applied to other major forms of stored carbon such as lipids.
Many aspects of plant architecture, developmental programs, and signal transduction are regulated by members of families of transcription factors such as MYBs and MYCs and MAD box proteins. The cauliflower mutant of Arabidopsis is one of many examples of alteration in expression of a transcription factor leading to a profound alteration in morphology and development (Kempin et al., 1995). One can envisage creating libraries of recombinant chimeras of transcription factors from these gene families and screening for desired changes in morphology or development. Such changes might include alterations in the amount and/or composition of cellulose for improved biomass accumulation.
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