For ~10,000 years, humans have been tailoring plants to meet their needs. The vast majority of this crop development occurred as a result of conventional breeding, that is, by recombining germplasm within the natural breeding barrier. The results were spectacular improvements in terms of output (harvestable) traits like yield, and to a lesser extent input (protective) traits such as disease resistance and stress tolerance. Recently, conventional breeding has been greatly enhanced by the development of molecular tools. A second wave of improvement occurred over the past 20 years or so with the development of methods of plant transformation of genes irrespective of source, with the use of techniques such as Agrobacterium tumefaciens-mediated transformation and DNA particle bombardment. In contrast to conventional breeding, the major impacts thus far have been with input traits such as insect and disease resistance. The introduction of engineered enzymes can be considered as a third wave of plant improvement in which enzymes with specific tailored properties are introduced into plants with the goal of conveying specific desired traits. The first example of this was the introduction of an engineered thioesterase from Garcinia mangostana into canola that resulted in increased accumulation of stearic acid (Facciotti et al., 1999).
With the emerging wealth of genome information, and the availability of genes from increasing numbers of organisms, one might ask why engineer genes instead of simply looking for naturally occurring genes that encode enzymes that already perform the desired transformation? The simplest answer is that a desired enzyme might not occur in any natural system. An example might be a biotransformation for which the substrate is a compound not normally found in nature. Second, one might identify an enzyme that performs the desired transformation, but does so very poorly. To make the enzyme useful, its activity would need to be optimized for the desired substrate. Third, the enzyme might have good in vitro activity, but may behave poorly in the metabolic context of the new host. Thus, the performance of the enzyme has the potential to be dramatically improved for use under a specific set of conditions. This could be the case if protein–protein interactions are necessary for function or if a particular concentration of cofactor is required. Enzyme engineering can modulate the Km for substrates and cosubstrates. Finally, the fold of the enzyme may present an inherent limitation to achieving the optimal catalytic rate for a desired biotransformation, and it might be better to start with a different protein fold that will allow a higher turnover to be achieved.
The goal of this chapter is to present the rationale for plant enzyme engineering in the context of improving plants to meet the increasing and changing demands of society. To achieve this, I will first lay a conceptual framework for understanding enzyme evolution as it occurs in nature and then show how the results of this process may not be ideal for transgenic applications. Next, I will describe approachesemployed for laboratory evolution of enzymes. Finally, I will summarize where I see future benefits and applications of these technologies.
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